The cytotoxic molecule granulysin is capable of inducing either chemotaxis or fugetaxis in dendritic cells depending on maturation: a role for Vδ2+ γδ T cells in the modulation of immune response to tumour?

Release of granulysin by γδ T cells contributes to tumour cell killing. A cytolytic 9000 MW isoform of granulysin kills tumour cells directly, whereas a 15 000 MW precursor has been hypothesized to cause both the maturation and migration of dendritic cell (DC) populations. Recruiting DC to a tumour is beneficial as these cells initiate adaptive immune responses, which contribute to the eradication of malignancies. In this study, Vδ2+ γδ T cells were activated by stimulation of peripheral blood mononuclear cells with zoledronic acid or Bacillus Calmette-Guérin (BCG), or were isolated and cultured with tumour targets. Although a large proportion of resting Vδ2+ γδ T cells expressed 15 000 MW granulysin, 9000 MW granulysin expression was induced only after stimulation with BCG. Increased levels of activation and granulysin secretion were also observed when Vδ2+ γδ T cells were cultured with the human B-cell lymphoma line Daudi. High concentrations of recombinant 15 000 MW granulysin caused migration and maturation of immature DC, and also initiated fugetaxis in mature DC. Conversely, low concentrations of recombinant 15 000 MW granulysin resulted in migration of mature DC, but not immature DC. Our data therefore support the hypothesis that Vδ2+ γδ T cells can release granulysin, which may modulate recruitment of DC, initiating adaptive immune responses.

Enhanced effect of checkpoint inhibitors when given after or together with IMM-101: significant responses in four advanced melanoma patients with no additional major toxicity.

BACKGROUND: The use of checkpoint inhibitors (ipilimumab, pembrolizumab, nivolumab) has revolutionised the treatment of metastatic melanoma. However still more than the half the patients do not respond to single-agent immunotherapy. This has led to the development of combining these agents in an attempt to enhance the anti-cancer activity. More than 300 different studies with 15 different drug doses are currently ongoing. Combining different checkpoint inhibitors (CPIs) does indeed lead to an increase in response rate, but this is associated with significant toxicity. IMM-101 is a heat killed Mycobacterium preparation which induces marked immune modulation and little systemic toxicity. It has been reported as having activity in melanoma as single agent and in pancreatic cancer in combination with gemcitabine, the latter in a randomised study. METHODS: Here we report the effect of adding CPIs to 3 patients who had previously been on IMM-101, either as a trial or a named patient programme and a patient who received the IMM-101 together with nivolumab. RESULTS: All 4 patients had rapid and very good responses, three of them maintained over 18 months with no significant additional toxicity. CONCLUSIONS: The rapid and complete clinical responses seen in these patients may suggest that IMM-101 is activating a complementary pathway which is synergistic with CPI treatment.

Zoledronic acid renders human M1 and M2 macrophages susceptible to Vδ2+ γδ T cell cytotoxicity in a perforin-dependent manner.

Vδ2+ T cells are a subpopulation of γδ T cells in humans that are cytotoxic towards cells which accumulate isopentenyl pyrophosphate. The nitrogen-containing bisphosphonate, zoledronic acid (ZA), can induce tumour cell lines to accumulate isopentenyl pyrophosphate, thus rendering them more susceptible to Vδ2+ T cell cytotoxicity. However, little is known about whether ZA renders other, non-malignant cell types susceptible. In this study we focussed on macrophages (Mϕs), as these cells have been shown to take up ZA. We differentiated peripheral blood monocytes from healthy donors into Mϕs and then treated them with IFN-γ or IL-4 to generate M1 and M2 Mϕs, respectively. We characterised these Mϕs based on their phenotype and cytokine production and then tested whether ZA rendered them susceptible to Vδ2+ T cell cytotoxicity. Consistent with the literature, IFN-γ-treated Mϕs expressed higher levels of the M1 markers CD64 and IL-12p70, whereas IL-4-treated Mϕs expressed higher levels of the M2 markers CD206 and chemokine (C-C motif) ligand 18. When treated with ZA, both M1 and M2 Mϕs became susceptible to Vδ2+ T cell cytotoxicity. Vδ2+ T cells expressed perforin and degranulated in response to ZA-treated Mϕs as shown by mobilisation of CD107a and CD107b to the cell surface. Furthermore, cytotoxicity towards ZA-treated Mϕs was sensitive-at least in part-to the perforin inhibitor concanamycin A. These findings suggest that ZA can render M1 and M2 Mϕs susceptible to Vδ2+ T cell cytotoxicity in a perforin-dependent manner, which has important implications regarding the use of ZA in cancer immunotherapy.

Randomised, open-label, phase II study of gemcitabine with and without IMM-101 for advanced pancreatic cancer.

BACKGROUND: Immune Modulation and Gemcitabine Evaluation-1, a randomised, open-label, phase II, first-line, proof of concept study (NCT01303172), explored safety and tolerability of IMM-101 (heat-killed Mycobacterium obuense; NCTC 13365) with gemcitabine (GEM) in advanced pancreatic ductal adenocarcinoma. METHODS: Patients were randomised (2 : 1) to IMM-101 (10 mg ml(-l) intradermally)+GEM (1000 mg m(-2) intravenously; n=75), or GEM alone (n=35). Safety was assessed on frequency and incidence of adverse events (AEs). Overall survival (OS), progression-free survival (PFS) and overall response rate (ORR) were collected. RESULTS: IMM-101 was well tolerated with a similar rate of AE and serious adverse event reporting in both groups after allowance for exposure. Median OS in the intent-to-treat population was 6.7 months for IMM-101+GEM v 5.6 months for GEM; while not significant, the hazard ratio (HR) numerically favoured IMM-101+GEM (HR, 0.68 (95% CI, 0.44-1.04, P=0.074). In a pre-defined metastatic subgroup (84%), OS was significantly improved from 4.4 to 7.0 months in favour of IMM-101+GEM (HR, 0.54, 95% CI 0.33-0.87, P=0.01). CONCLUSIONS: IMM-101 with GEM was as safe and well tolerated as GEM alone, and there was a suggestion of a beneficial effect on survival in patients with metastatic disease. This warrants further evaluation in an adequately powered confirmatory study.

Human Peritoneal Mesothelial Cells Display Phagocytic and Antigen-Presenting Functions to Contribute to Intraperitoneal Immunity.

Mesothelial cells lining the peritoneal cavity are strategically positioned to respond to and counter intraperitoneal infections, cancer cells, and other challenges. We have investigated human peritoneal mesothelial cells (HPMCs) for phagocytic activity, expression of surface Major Histocompatibility Complex (MHC) class II and accessory molecules involved in antigen presentation, and the ability to present recall antigens to T cells. Phagocytosis of dextran, latex beads, and Escherichia coli was observed by flow cytometry, and internalization was visualized using confocal and electron microscopy. Flow cytometry and/or cellular enzyme-linked immunosorbent assay showed constitutive expression of ICAM-1, LFA-3, and B7-1, but not B7-2 or MHC class II. Interferon-gamma induced MHC II and ICAM-1 expression in a dose- and time-dependent manner. Importantly, HPMCs induced autologous CD3 T-lymphocyte proliferation (H incorporation) after pulse with recall antigen. Human peritoneal mesothelial cells equipped with phagocytic and antigen-presenting machinery are anticipated to have an integral role in intraperitoneal immune surveillance.

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.

Enhanced cross-priming of naive CD8+ T cells by dendritic cells treated by the IMiDs® immunomodulatory compounds lenalidomide and pomalidomide.

The IMiDs(®) immunomodulatory compounds lenalidomide and pomalidomide are agents with anti-inflammatory, immunomodulatory and anti-cancer activity. An excellent success rate has been shown for multiple myeloma in phase I/II clinical trials leading to Food and Drug Administration approval of lenalidomide. One mechanism by which these drugs could enhance anti-tumour immunity may be through enhanced dendritic cell (DC) function. Thalidomide, a compound structurally related to lenalidomide and pomalidomide, is known to enhance DC function, and we have investigated whether its analogues, pomalidomide and lenalidomide, also have functional effects on DCs. We used mouse bone marrow-derived DCs treated with 5 or 10 µm pomalidomide, or lenalidomide from day 1 of culture. Treatment with IMiD(®) immunomodulatory compounds increased expression of Class I (H2-Kb), CD86, and pomalidomide also increased Class II (I-Ab) expression in bone marrow-derived DCs, as measured by flow cytometry. Fluorescent bead uptake was increased by up to 45% when DCs were treated with 5 or 10 µm pomalidomide or lenalidomide compared with non-treated DCs. Antigen presentation assays using DCs primed with ovalbumin, and syngeneic T cells from transgenic OTI and OTII mice (containing MHC restricted, ovalbumin-specific, T cells) showed that both pomalidomide and lenalidomide effectively increased CD8(+) T-cell cross-priming (by up to 47%) and that pomalidomide alone was effective in increasing CD4(+) T-cell priming (by 30%). Our observations suggest that pomalidomide and lenalidomide enhance tumour antigen uptake by DCs with an increased efficacy of antigen presentation, indicating a possible use of these drugs in DC vaccine therapies.

Analysis of surrogate gene expression markers in peripheral blood of melanoma patients to predict treatment outcome of adjuvant pegylated interferon alpha 2b (EORTC 18991 side study).

We analysed mRNA levels of interferon response genes (ISG15, STAT1, CXCL10) of inhibitors of the JAK/STAT pathway (STAT3, SOCS1, SOCS3) and of cytokines (TNFα, IL10, TGFß1) in peripheral blood of 91 stage III melanoma patients enrolled in EORTC 18991 trial to find biomarkers indicative for disease stage and predictive for efficacy of pegylated interferon alpha-2b (PEG-IFNα-2b) therapy. mRNA levels were analysed at baseline and after 6 months. Univariate and multivariate analyses were performed to estimate the prognostic and predictive role of mRNA levels for distant metastasis-free survival (DMFS) and relapse-free survival (RFS). Compared to healthy controls, melanoma patients showed significantly higher TGFß1 mRNA levels. In a multivariate model, increasing SOCS1 and SOCS3 mRNA levels were associated with worse RFS (P = 0.02 and P = 0.04, respectively) and DMFS (P = 0.05 and P = 0.05, respectively) due to negative correlation between, respectively, SOCS1/SOCS3 mRNA levels and ulceration or Breslow thickness. No impact of PEG-IFNα-2b on mRNA levels was observed except for ISG15 mRNA levels, which decreased in the treatment arm (P = 0.001). It seems that patients with a decrease >60 % of ISG15 mRNA levels during 6 months PEG-IFNα-2b had inferior outcome.

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(+) αß 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.

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.

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 T(H)1 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-1ß 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.

The role of immune modulation and anti-inflammatory agents in the management of prostate cancer: A case report of six patients.

Cancer is associated with chronic inflammation and disruption to normal immune function. As such, the ability to thrive in a chronically inflamed microenvironment is regarded as a hallmark of cancer. Therefore, targeting inflammation and/or correction of aberrant immunity has been a therapeutic aim. The aim of the present study was to describe the use of a novel immunotherapy, called IMM-101, which is a naturally occurring, heat-killed whole cell mycobacterium, used in combination with conventional treatments in patients with prostate cancer. The present study analysed and presented data from six patients diagnosed with prostate cancer, some of whom have metastatic disease. Treatment regimens included the use of IMM-101, the correction of vitamin D3 levels, and combination with other agents that have anti-inflammatory and immune-modulatory abilities, such as bromelain and low-dose naltrexone (LDN). Clinical responses were detected in the patients when IMM-101 was commenced and further improvements were seen when an anti-inflammatory agent was used in unison. Combination therapy quickly led to a reduction in prostate-specific antigen levels, and stabilisation of disease was often achieved as indicated by repeat MRI and PET scans. Few side effects of any kind were observed when using these combination treatments. In conclusion, IMM-101 treatment alongside an anti-inflammatory agent, such as bromelain and/or LDN, may be considered an active and safe drug combination, and is a regimen that should be considered for treating patients with prostate cancer.

Naltrexone at low doses (LDN) and its relevance to cancer therapy.

INTRODUCTION: Naltrexone was designed to inhibit opioid receptors without activating them and hence used to block the stimulatory effects of morphine and heroin. It was noted that in certain patients being treated with naltrexone for an opioid addiction many reported significant secondary benefit when being weaned off naltrexone. This group of patients had chronic inflammatory and autoimmune conditions and reported improvements whilst using the lower dosages of naltrexone. There have also been recent anecdotal reports of cancer resolution following the use of low doses of naltrexone (LDN). However, the mechanism of action is unclear. AREAS COVERED: We review three mechanisms through which LDN can influence cancer progression; namely, (a) antagonism of receptors to which LDN binds, which include toll-like receptors 7-9 that lead to IL-6 suppression b) modulation of immune function in patients; and c) direct inhibition of signaling pathways involved in cancer cell control, including the priming of pro-apoptotic pathways. EXPERT OPINION: Considering the increase in the number of anecdotal reports of activity, there will likely be a bigger drive toward using LDN in the oncological setting. These reports support clinical trials of LDN in cancer, especially when given in combination with certain chemotherapy.

Combination of cannabidiol with low­dose naltrexone increases the anticancer action of chemotherapy in vitro and in vivo.

We previously reported that both cannabidiol (CBD) and low­dose naltrexone (LDN) exhibit complex effects on G­protein coupled receptors, which can impact the expression and function of other members of this superfamily. These receptors feed into and interact with central signalling cascades that determine the ease by which cells engage in apoptosis, and can be used as a way to prime cancer cells to other treatments. The present study was designed to investigate the effect of combining these two agents on cancer cell lines in vitro and in a mouse model, and focused on how the sequence of administration may affect the overall action. The results showed both agents had minimal effect on cell numbers when used simultaneously; however, the combination of LDN and CBD, delivered in this specific sequence, significantly reduced the number of cells, and was superior to the regimen where the order of the agents was reversed. For example, there was a 35% reduction in cell numbers when using LDN before CBD compared to a 22% reduction when using CBD before LDN. The two agents also sensitised cells to chemotherapy as significant decreases in cell viability were observed when they were used before chemotherapy. In mouse models, the use of both agents enhanced the effect of gemcitabine, and crucially, their use resulted in no significant toxicity in the mice, which actually gained more weight compared to those without this pre­treatment (+6.5 vs. 0%). Overall, the results highlight the importance of drug sequence when using these drugs. There is also a need to translate these observations into standard chemotherapy regimens, especially for common tumour types where treatment is often not completed due to toxicities.

The antimalarial agent artesunate possesses anticancer properties that can be enhanced by combination strategies.

Artemisinins are a class of compounds that are first-line treatment options for malaria. They also have potent antiproliferative activity, which makes them potential anticancer drugs. We have previously demonstrated anticancer activity of a number of these compounds in vitro; however, cytotoxic activities were compromised by drug-induced cell cycle arrests. In this study, we have explored further the activity of the clinical lead artemisinin-drug artesunate (ART), used either alone or in combination with established chemotherapy. Also, by using a cell line expressing polyploidy character, have also explored the impact of cell cycle arrest in determining overall drug activity. Results showed that ART caused dose-dependent decreases in cell number, which were associated with either increased cytotoxicity or cytostasis. Cytostasis appeared to be a consequence of a simultaneous arrest at all phases of the cell cycle, a deduction that was supported by molecular profiling, which showed reductions in cell cycle transit proteins. ART appeared to maintain cells in this arrested state; however, reculturing these treated cells in drug-free medium resulted in significant reductions in viabilities. We also showed that ART maintained activity in polyploidy cells, and that an impressive enhancement to its activity was achievable through a combination with the immunomodulatory drug lenalidomide. Taken together, these observations indicate ART and its related compounds may be effective for the treatment of tumours, and that activity is related to schedule. Therefore, it is important to carefully select the most appropriate schedule to maximise ART efficacy.

The immuno-oncological implications of insulin.

Emerging evidence has implicated insulin in regulating the phenotypes of various immune cells through canonical downstream signalling effectors of insulin, namely, the PI3K/Akt/mTOR pathway. Notably, these signalling components also exhibit crosstalk with other immune signalling pathways, such as the JAK/STAT pathway (activated by cytokines and growth factors), and, importantly, are also negatively regulated by the immune checkpoint blockers (ICBs), PD-1 and CTLA-4. Here, we point out recent findings, suggesting that insulin may promote a pro-inflammatory phenotype with potential implications on ICB therapy. As an example, the contemporary paradigm holds that, while T cell receptor recognition of distinct MHC-expressed epitopes ensures specificity, co-activation of CD28 along with signal inputs form various cytokines and insulin operates to 'fine-tune' the immune response via PI3K and other downstream signalling molecules. These considerations highlight the urgent need for focused investigations into the role of insulin in regulating immune cell function in the context of ICB therapies.

A microarray study of altered gene expression in colorectal cancer cells after treatment with immunomodulatory drugs: differences in action in vivo and in vitro.

Thalidomide and lenalidomide are FDA approved for the treatment of multiple myeloma, and along with pomalidomide are being investigated in a variety of other cancers. Although these agents display immunomodulatory, anti-angiogenic and anti-apoptotic effects, little is known about the primary mode of therapeutic action in patients with cancer. This paper describes a microarray study of the in vitro and in vivo effects of these drugs, and contrasts the difference in gene profiles achieved in the two models. In the current study, Agilent whole mouse genome oligonucleotide microarrays (44 K) were used to examine alterations in gene expression of colorectal cancer cells after treatment. Venn analysis revealed a divergence of gene signature for pomalidomide and lenalidomide, which although similar in vitro, different in vivo. Several clusters of genes involved in various cellular processes such as immune response, cell signalling and cell adhesion were altered by treatment, and common to the three drugs. Notably, the expressions of linked genes within the Notch/Wnt signalling pathway, including kremen2 and dtx4, highlighted a possible novel mechanistic pathway for these drugs. This study also showed that gene signatures were not greatly divergent in the models, and recapitulated the complex nature of these drugs. Overall, these microarray studies highlighted the diversity of this class of drug, which have effects ranging from cell signalling to translation initiation.

Long-term benefit from immune modulation and anti-inflammatory treatment in metastatic mesothelioma.

A 64 year old male heating engineer was investigated for a persistent cough and found to have epithelioid mesothelioma with pleural effusion, lung nodules and increased thoracic lymph nodes. He declined standard of care treatment following his own research and he was enrolled in a named patient programme of IMM-101. He was advised to correct his low vitamin D3 level and to start using anti-inflammatories such as aspirin, bromelain and low dose Naltrexone. At review one year later a CT scan showed no change and he continued on the regimen. Four years after the diagnosis a CT scan showed that there was a modest but definite progression of the left malignant pleural thickening, and a new right-sided effusion, enlargement of several intrathoracic nodes which had been noted on the early scans. The chest wall lump eventually broke down and required local radiotherapy. He then developed abdominal pain and found to have peritoneal disease. Last year he obtained the cannabinoids CBD and THC which slowed down the disease and a CT scan after he had been on this for six months, showed that his disease was fairly stable with marginal progression.