Dominant immunosuppression of dendritic cell function by prostate-cancer-derived exosomes

Exosomes are a distinct population of extracellular vesicles of endocytic origin with a protein repertoire similar to the parent cell. Although tumour-derived exosomes harbour immunosuppressive characteristics, they also carry tumour antigens and thus potentially contribute to immune activation. The aim of this study was to examine the impact of prostate cancer exosomes on tumour antigen cross-presentation. DU145 cells, transduced with shRNA to knockdown Rab27a (DU145KD) that inhibits exosome secretion, triggered significantly stronger tumour-antigen-specific T cell responses when loaded onto dendritic cells (DC) than control DU145 cells. Enhanced T cell response was prevented by adding purified exogenous DU145 exosomes to DU145KD cells, demonstrating that the dominant effect of tumour exosomes is immunosuppression and not antigen delivery. CD8+ T cell responses were impaired via exosomal regulation of DC function; exosomes triggered the expression of CD73, an ecto-5-nucleotidase responsible for AMP to adenosine hydrolysis, on DC. CD73 induction on DC that constitutively express CD39 resulted in an ATP-dependent inhibition of TNFα- and IL-12-production. We identified exosomal prostaglandin E2 (PGE2) as a potential driver of CD73 induction, as inhibition of PGE2 receptors significantly reduced exosome-dependent CD73 induction. The results reveal a hitherto unknown suppression of DC function via exosomal PGE2, adding a new element to tumour exosome–immune cell cross-talk

Prostaglandin E2-mediated adenosinergic effects on CD14+ cells: self-amplifying immunosuppression in cancer

CD39 and CD73 are surface-expressed ectonucleotidases that hydrolyze ATP in a highly regulated, serial manner into ADP, AMP and adenosine. The end product, adenosine, has both tumor-promoting and immunosuppressive effects. The aim of this study was to determine CD73 expression on immune cells in pleural effusion (PE) in order to have a better understanding of the immune environment in mesothelioma. PE- or blood-derived CD14+ cells of mesothelioma patients and healthy donors were analyzed by flow cytometry for the expression of CD39 and CD73. CD73-induction was studied by exposure of CD14+ cells to the soluble fraction of PE (sPE), while the signaling mechanism, responsible for CD73 induction, by phosphoflow cytometry and receptor-inhibition studies. We observed CD73 expression on CD14+ cells in PE but not peripheral blood of mesothelioma patients or healthy donors. CD73 expression was inducible on CD14+ cells with sPE, cyclic-AMP (cAMP)-inducers (forskolin and prostaglandin-E2 (PGE2)) and adenosine. Inhibition of PGE2 receptors or adenosine A2 receptors blocked CD73-induction by sPE. sPE treatment triggered protein kinase A and p38 activation. However, signal-transducer and activator of transcription 3 (STAT3)-blocking led to enhanced CD73 expression, demonstrating a hitherto unknown negative control of purinergic signaling by STAT3 in CD14+ cells. TNFα production by CD73+ CD14+ cells was significantly impaired in the presence of AMP, confirming immunosuppressive function. Taken together, CD73 expression can be induced by PGE2, cAMP or adenosine on human CD14+ cells. We suggest that targeting this autocrine loop is a valid therapeutic approach in mesothelioma that may also enhance immunotherap

Overexpression and potential targeting of the oncofoetal antigen 5T4 in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is resistant to conventional treatments. Novel, targeted treatments are hampered by the relative lack of MPM-associated tumour antigens. The aim of this study was to evaluate the level of expression and the relevance of 5T4 as a tumour-associated antigen in MPM. 5T4 expression was assessed by Western blotting, flow cytometry, immuno-cytochemistry and -histochemistry in 11 mesothelioma cell lines, 21 tumour biopsies, and ex vivo tumour cells obtained from the pleural fluid (PF) of 10 patients. 5T4 antibody levels were also determined in the plasma of patients and healthy donors. The susceptibility of MPM cells to 5T4-specific T-cell-mediated killing was determined using an HLA-A2+, CD8+ T-cell line, developed against the 5T417–25 peptide. We report here that cell surface 5T4 expression was detected in all mesothelioma cell lines and PF cell samples. Mesothelin and CD200, a suggested mesothelioma marker, were co-expressed with 5T4 on tumour cells in PF. Immunohistochemistry confirmed overexpression of 5T4, similar to mesothelin, on tumour cells but not on reactive stroma in all tissue sections tested. Median 5T4 antibody levels were 46% higher in patient than in healthy donor plasma, indicating immune recognition. Importantly, 5T4-specific CD8+ T-cells were able to kill four out of six HLA-A2+ MPM cell lines but not an HLA-A2− cell line, demonstrating immune recognition of MPM-associated 5T4 antigen at the effector T-cell level. We conclude that 5T4 is a potential new antigen for targeted therapies such as immunotherapy in MPM, as it is overexpressed on mesothelioma cells and recognised by 5T4-specific cytotoxic T-cells. Our findings have been translated into a Phase II clinical trial applying 5T4-targeted therapies in MPM patients

Tumour antigen cross-presentation from irradiated tumour cells and the role of tlr4 polymorphism

Immune responses contribute to the success of radiation therapy of solid tumours; however, the mechanism of triggering CD8+ T cell responses is poorly understood. Antigen cross-presentation from tumour cells by dendritic cells (DC) is a likely dominant mechanism to achieve CD8+ T cell stimulation. We established a cross-presentation model in prostate cancer in which DC present a naturally expressed oncofetal tumour antigen (5T4) from irradiated DU145 tumour cells to 5T4-specific T cells. Ionising radiation (12 Gy) caused G2/M cell cycle arrest and cell death, increased cellular 5T4 and high-mobility protein group-B1 (HMGB1) levels and upregulated surface calreticulin and Hsp70 expression in DU145 cells. Co-culture of DC with irradiated tumour cells lead to efficient phagocytosis of tumour cells and upregulation of CD86 and HLA-DR on DC. CD8+ 5T4-specific T cells, stimulated with these DC, proliferated and produced IFNγ. Inhibition of HMGB1 decreased T cell stimulation but not DC activation, while TRIF/MyD88 inhibition only had a marginal effect on T cell stimulation. Unlike previous reports, I found no functional evidence that DC with Asp299Gly toll-like receptor-4 (TLR4) single nucleotide polymorphism had impaired ability to cross-present tumour antigen. However, I observed a highly significant and robust prevention of antigen cross-presentation when tumour cells were pretreated with the novel Hsp70 inhibitor, VER 155008. The inhibitor also prevented CD86 upregulation on DC co-cultured with irradiated tumour cells. Together, the results in this thesis demonstrate that radiation induces immunologically relevant changes in tumour cells, which can trigger CD8+ T cell responses via a predominantly Hsp70-dependent antigen cross-presentation process

Tumor stroma-derived factors skew monocyte to dendritic cell differentiation toward a suppressive CD14+PD-L1+phenotype in prostate cancer

Tumor-associated stromal myofibroblasts are essential for the progression and metastatic spread of solid tumors. Corresponding myeloid cell infiltration into primary tumors is a negative prognostic factor in some malignancies. The aim of this study was to define the exact role of stromal myofibroblasts and stromal factors in early prostate carcinoma (PCa) regulating monocyte infiltration and differentiation into dendritic cells (DCs). Epithelial and stromal primary cultures were generated from PCa biopsies and their purity confirmed. Stromal cells produced significantly more of the (C‒C) motif chemokine ligand 2 (CCL2), interleukin 6 (IL-6) and transforming growth factor β (TGFβ) than epithelial cells. Monocyte chemoattraction was predominantly due to stromal-derived factors, mainly CCL2. DCs generated in the presence of stromal (but not epithelial) factors upregulated CD209, but failed to downregulate the monocyte marker CD14 in a signal transducer and activator of transcription 3 (STAT3)-dependent manner. Monocytes exposed to stromal factors did not produce detectable amounts of IL-10, however, upon lipopolysaccharide stimulation, stromal factor generated dendritic cells (sDC) produced significantly more IL-10 and less IL-12 than their conventional DC counterparts. sDC failed to cross-present tumor-antigen to CD8+ T cells and suppressed T-cell proliferation. Most importantly, sDC expressed significantly elevated levels of programmed cell death ligand-1 (PD-L1) in a primarily STAT3 and IL-6-dependent manner. In parallel with our findings in vitro, tumor-infiltrating CD14+ cells in situ were found to express both PD-L1 and CD209, and a higher percentage of tumor-associated CD3+ T cells expressed programmed cell death-1 (PD-1) molecules compared to T cells in blood. These results demonstrate a hitherto undescribed, fundamental contribution of tumor-associated stromal myofibroblasts to the development of an immunosuppressive microenvironment in early PCa

Resistance to HER2-targeted anti-cancer drugs is associated with immune evasion in cancer cells and their derived extracellular vesicles

Neuromedin U (NmU) -a neuropeptide belonging to the neuromedin family? plays a substantial role in HER2-positive breast cancer, correlating with increased aggressiveness, resistance to HER2-targeted therapies and overall significantly poorer outcome for patients. However, the mechanism through which it exerts these effects remains unclear. To elucidate this, initially we used HER2-positive breast cancer cells stably over-expressing NmU. These cells and their released extracellular vesicles (EVs) had increased amounts of the immunosuppressive cytokine TGF?1 and the lymphocyte activation inhibitor PD-L1. Furthermore, these cells also showed enhanced resistance to antibody-dependent cell cytotoxicity (ADCC) mediated by trastuzumab, indicating a role of NmU in enhancing immune evasion. All these features were also found in HER2-targeted drug-resistant cells which we previously found to express higher levels of NmU than their drug-sensitive counterparts. Interestingly, EVs from drug-resistant cells were able to increase levels of TGF?1 in drug-sensitive cells. In our neo-adjuvant clinical trial, TGF?1 levels were significantly higher in EVs isolated from the serum of patients with HER2-overexpressing breast cancers who went on to not respond to HER2-targeted drug treatment, compared with those who experienced complete or partial response. Taken together, our results report a new mechanism-of-action for NmU in HER2-overexpressing breast cancer that enhances resistance to the anti-tumor immune response. Furthermore, EV levels of TGF?1 correlating with patients\u27 response versus resistance to HER2-targeted drugs suggests a potential use of EV-TGF?1 as a minimally-invasive companion diagnostic for such treatment in breast cancer

Cross-Presentation of the Oncofetal Tumor Antigen 5T4 from Irradiated Prostate Cancer Cells—A Key Role for Heat-Shock Protein 70 and Receptor CD91

Immune responses contribute to the success of radiotherapy of solid tumors; however, the mechanism of triggering CD8+ T-cell responses is poorly understood. Antigen cross-presentation from tumor cells by dendritic cells (DC) is a likely dominant mechanism to achieve CD8+ T-cell stimulation. We established a cross-presentation model in which DCs present a naturally expressed oncofetal tumor antigen (5T4) from irradiated DU145 prostate cancer cells to 5T4-specific T cells. The aim was to establish which immunogenic signals are important in radiation-induced cross-presentation. Radiation (12 Gy) caused G2–M cell-cycle arrest and cell death, increased cellular 5T4 levels, high-mobility protein group-B1 (HMGB1) release, and surface calreticulin and heat-shock protein-70 (Hsp70) expression in DU145 cells. DCs phagocytosed irradiated tumor cells efficiently, followed by upregulation of CD86 on phagocytic DCs. CD8+ 5T4-specific T cells, stimulated with these DCs, proliferated and produced IFNγ. Inhibition of HMGB1 or the TRIF/MyD88 pathway only had a partial effect on T-cell stimulation. Unlike previous investigators, we found no evidence that DCs carrying Asp299Gly Toll-like receptor-4 (TLR4) single-nucleotide polymorphism had impaired ability to cross-present tumor antigen. However, pretreatment of tumor cells with Hsp70 inhibitors resulted in a highly statistically significant and robust prevention of antigen cross-presentation and CD86 upregulation on DCs cocultured with irradiated tumor cells. Blocking the Hsp70 receptor CD91 also abolished cross-presentation. Together, the results from our study demonstrate that irradiation induces immunologically relevant changes in tumor cells, which can trigger CD8+ T-cell responses via a predominantly Hsp70-dependent antigen cross-presentation process

Enhancement of T cell responses as a result of synergy between lower doses of radiation and T cell stimulation

As a side effect of cancer radiotherapy, immune cells receive varying doses of radiation. Whereas high doses of radiation (>10 Gy) can lead to lymphopenia, lower radiation doses (2–4 Gy) represent a valid treatment option in some hematological cancers, triggering clinically relevant immunological changes. Based on our earlier observations, we hypothesized that lower radiation doses have a direct positive effect on T cells. In this study, we show that 0.6–2.4 Gy radiation enhances proliferation and IFN-γ production of PBMC or purified T cells induced by stimulation via the TCR. Radiation with 1.2 Gy also lowered T cell activation threshold and broadened the Th1 cytokine profile. Although radiation alone did not activate T cells, when followed by TCR stimulation, ERK1/2 and Akt phosphorylation increased above that induced by stimulation alone. These changes were followed by an early increase in glucose uptake. Naive (CD45RA+) or memory (CD45RA−) T cell responses to stimulation were boosted at similar rates by radiation. Whereas increased Ag-specific cytotoxic activity of a CD8+ T cell line manifested in a 4-h assay (10–20% increase), highly significant (5- to 10-fold) differences in cytokine production were detected in 6-d Ag-stimulation assays of PBMC, probably as a net outcome of death of nonstimulated and enhanced response of Ag-stimulated T cells. T cells from patients receiving pelvic radiation (2.2–2.75 Gy) also displayed increased cytokine production when stimulated in vitro. We report in this study enhanced T cell function induced by synergistic radiation treatment, with potential physiological significance in a wide range of T cell responses

Enhancement of T Cell Responses as a Result of Synergy between Lower Doses of Radiation and T Cell Stimulation

As a side effect of cancer radiotherapy, immune cells receive varying doses of radiation. Whereas high doses of radiation (>10 Gy) can lead to lymphopenia, lower radiation doses (2–4 Gy) represent a valid treatment option in some hematological cancers, triggering clinically relevant immunological changes. Based on our earlier observations, we hypothesized that lower radiation doses have a direct positive effect on T cells. In this study, we show that 0.6–2.4 Gy radiation enhances proliferation and IFN-γ production of PBMC or purified T cells induced by stimulation via the TCR. Radiation with 1.2 Gy also lowered T cell activation threshold and broadened the Th1 cytokine profile. Although radiation alone did not activate T cells, when followed by TCR stimulation, ERK1/2 and Akt phosphorylation increased above that induced by stimulation alone. These changes were followed by an early increase in glucose uptake. Naive (CD45RA+) or memory (CD45RA−) T cell responses to stimulation were boosted at similar rates by radiation. Whereas increased Ag-specific cytotoxic activity of a CD8+ T cell line manifested in a 4-h assay (10–20% increase), highly significant (5- to 10-fold) differences in cytokine production were detected in 6-d Ag-stimulation assays of PBMC, probably as a net outcome of death of nonstimulated and enhanced response of Ag-stimulated T cells. T cells from patients receiving pelvic radiation (2.2–2.75 Gy) also displayed increased cytokine production when stimulated in vitro. We report in this study enhanced T cell function induced by synergistic radiation treatment, with potential physiological significance in a wide range of T cell responses