Ian Daniels

Citrullinated Vimentin Presented on MHC-II in Tumor Cells Is a Target for CD4+ T-Cell–Mediated Antitumor Immunity

Stressful conditions in the harsh tumor microenvironment induce autophagy in cancer cells as a mechanism to promote their survival. However, autophagy also causes post-translational modification of proteins that are recognized by the immune system. In particular, modified self-antigens can trigger CD4(+) T-cell responses that might be exploited to boost antitumor immune defenses. In this study, we investigated the ability of CD4 cells to target tumor-specific self-antigens modified by citrullination, which converts arginine residues in proteins to citrulline. Focusing on the intermediate filament protein vimentin, which is frequently citrullinated in cells during epithelial-to-mesenchymal transition of metastasizing epithelial tumors, we generated citrullinated vimentin peptides for immunization experiments in mice. Immunization with these peptides induced IFNγ- and granzyme B-secreting CD4 T cells in response to autophagic tumor targets. Remarkably, a single immunization with modified peptide, up to 14 days after tumor implant, resulted in long-term survival in 60% to 90% of animals with no associated toxicity. This antitumor response was dependent on CD4 cells and not CD8(+) T cells. These results show how CD4 cells can mediate potent antitumor responses against modified self-epitopes presented on tumor cells, and they illustrate for the first time how the citrullinated peptides may offer especially attractive vaccine targets for cancer therapy.

A requirement for calcium in the caspase-independent killing of Burkitt lymphoma cell lines by Rituximab

The therapeutic monoclonal antibody rituximab has previously been shown to kill B cells in a caspase‐independent manner. The signalling pathways underpinning this novel death pathway are unknown. The present study showed that rituximab treatment of Burkitt lymphoma cell lines induced a slow rise in intracellular calcium ([Ca2+]i). This rise was only witnessed in cell lines that were killed by antibody, suggesting a critical role for Ca2+ in mediating rituximab‐driven caspase‐independent cell death. Inhibition of the two main intracellular store‐located Ca2+ channels, i.e. the ryanodine and inositol‐1,4,5‐triphosphate receptor channels by dantrolene and xestospongen‐c respectively did not prevent the rise in Ca2+ seen with rituximab or protect cells from subsequent death. In sharp contrast, inhibition of Ca2+ entry via plasma membrane channels with (2‐aminoethoxy) diphenylborate or SKF‐96365 or complete chelation of extracellular Ca2+ with ethyleneglycol bis (aminoethylether) tetra‐acetate inhibited the rise in [Ca2+]i and increased cell viability. Together, these data suggest that ligation of the CD20 receptor with rituximab allows a slow sustained influx of Ca2+ from the external environment that under certain conditions can lead to cell death.

Impaired Polymorphonuclear Neutrophil Function in End-Stage Renal Failure and Its Correction by Continuous Ambulatory Peritoneal Dialysis

The polymorphonuclear neutrophil (PMN) function of 22 patients with end-stage renal failure (ESRF) was studied immediately before and 3 months after starting continuous ambulatory peritoneal dialysis (CAPD) and compared with a control group of healthy normal volunteers. The PMN functions studied were phagocytosis and killing of Staphylococcus epidermidis and respiratory burst activity. The results show that in the presence of normal pooled human serum PMN from patients with ESRF before CAPD treatment phagocytose bacteria normally, but have impaired killing. Before treatment, the PMN from patients with ESRF also showed an increase in both unstimulated and stimulated superoxide anion production. Abnormal PMN function was corrected by CAPD treatment, suggesting the involvement of a dialyzable toxin.

Effect of Peritoneal Dialysis Effluent on Superoxide Anion Production by Polymorphonuclear Neutrophils

Peritoneal dialysis effluent (PDE) contains at least 2 factors capable of affecting superoxide generation by polymorphonuclear neutrophils (PMN) in response to both particulate and soluble stimuli. A low molecular weight fraction (< 1.2 kD) enhanced the response to the chemotactic peptide fMLP and to preopsonised Staphylococcus epidermidis and Candida guilliermondii. A higher molecular weight fraction (> 1.2 kD) inhibited superoxide production in response to phorbol myristate acetate (PMA). The effects of PDE were dose dependent over the range of 10-70% (v/v) and simply augmented and reduced the dose-response curve to fMLP and PMA, respectively. There was no alteration in the concentration of stimulus required to give maximal superoxide production in either case. These data suggest that factors capable of affecting superoxide production by PMN accumulate in uraemia and are removed from the circulation into dialysis fluid.

Impaired Bacterial Killing and Hydrogen Peroxide Production by Polymorphonuclear IMeutrophils in End-Stage Renal Failure

Polymorphonuclear leukocytes (PMN) isolated from a sub-population of patients with end-stage renal failure (ESRF) who were identified because they demonstrated impaired intracellular bacterial killing, were assayed for hydrogen peroxide activity using two different techniques capable of distinguishing between total and intracellular hydrogen peroxide generation. In an attempt to elucidate the mechanism of impaired intracellular bacterial killing further, hydrogen peroxide activity was compared to PMN isolated from patients receiving continuous ambulatory peritoneal dialysis and a control group of healthy normal volunteers. PMN from conservatively treated ESRF patients demonstrated reduced production of intracellular hydrogen peroxide (mean 37.7 +/- 4.3 fluorescence units), compared to PMN from both ESRF patients treated with continuous ambulatory peritoneal dialysis (mean 57.9 +/- 6.6 fluorescence units) and normal controls (mean 60.4 +/- 3.5 fluorescence units). This suggests that the probable mechanism of impaired bacterial intracellular killing by the PMN of conservatively treated ESRF patients involves the production of intracellular hydrogen peroxide.

Effect of a gluten‐free diet on plasma nitric oxide products in coeliac disease

Background: Inducible nitric oxide synthase is expressed in the small intestine of patients with coeliac disease. This produces increased plasma concentration of nitric oxide end products (NOx), most marked in those ingesting gluten. The time‐course of change in NOx with a gluten‐free diet (GFD) and its correlation with histology and coeliac serology were studied. Methods: Fasting plasma NOx was determined by the Greiss reaction in 20 coeliac patients at diagnosis and 2, 4 and 6 months after commencing a GFD. Endomysial and gliadin antibodies were checked at the same time. Duodenal biopsies were taken at diagnosis and at 6 months, and then graded according to the Marsh classification. Results: Plasma NOx fell rapidly following the introduction of a GFD (mean before GFD 95.8 μM to 61.5 μM at 2 months), and further still by 6 months (mean = 37.0 μM). Reductions at 2 and 6 months were statistically significant compared with baseline (P < 0.01 and P < 0.005, respectively: Wilcoxon signed ranks test). Plasma NOx was correlated with histological grade initially (P = 0.03: Kruskal‐Wallis) but not after 6 months on a GFD (P = 0.24). Coeliac serology correlated poorly with histology. Conclusions: Plasma NOx falls rapidly following GFD in coeliac disease and is related to histological grade initially. However, values vary widely between individuals, which may limit its use as a clinical tool.

Targeting gp100 and TRP-2 with a DNA vaccine: Incorporating T cell epitopes with a human IgG1 antibody induces potent T cell responses that are associated with favourable clinical outcome in a phase I/II trial

ABSTRACT A DNA vaccine, SCIB1, incorporating two CD8 and two CD4 epitopes from TRP-2/gp100 was evaluated in patients with metastatic melanoma. Each patient received SCIB1 via intramuscular injection with electroporation. The trial was designed to find the safest dose of SCIB1 which induced immune/clinical responses in patients with or without tumour. Fifteen patients with tumor received SCIB1 doses of 0.4-8 mg whilst 20 fully-resected patients received 2–8 mg doses. Twelve patients elected to continue immunization every 3 months for up to 39 months. SCIB1 induced dose-dependent T cell responses in 88% of patients with no serious adverse effects or dose limiting toxicities. The intensity of the T cell responses was significantly higher in patients receiving 4 mg doses without tumor when compared to those with tumor (p < 0.01). In contrast, patients with tumor showed a significantly higher response to the 8 mg dose than the 4 mg dose (p < 0.03) but there was no significant difference in the patients without tumor. One of 15 patients with measurable disease showed an objective tumor response and 7/15 showed stable disease. 5/20 fully-resected patients have experienced disease recurrence but all remained alive at the cut-off date with a median observation time of 37 months. A positive clinical outcome was associated with MHC-I and MHC-II expression on tumors prior to therapy (p = 0.027). We conclude that SCIB1 is well tolerated and stimulates potent T cell responses in melanoma patients. It deserves further evaluation as a single agent adjuvant therapy or in combination with checkpoint inhibitors in advanced disease.

Citrullinated α-enolase is an effective target for anti-cancer immunity

ABSTRACT Targeting post-translationally modified epitopes may provide a new strategy for generating tumor specific immune responses. Citrullination is the post-translational modification of arginine to citrulline catalyzed by peptidylarginine deaminase (PAD) enzymes. Presentation of citrullinated peptides on MHC-II has been associated with autophagy. Tumors upregulate autophagy and present citrullinated peptides in response to stresses including nutrient deprivation, oxygen deprivation, redox stress and DNA damage, making them good targets for immune attack. The ubiquitous glycolytic enzyme α-enolase (ENO1) is often citrullinated and degraded during autophagy. Immunization of mice with two citrullinated ENO1 peptides (ENO1 241–260cit253 or 11–25cit15) induced strong Th1 responses that recognize the post-translationally modified, but not the wild type unmodified epitope. ENO1 11–25cit15 induced tumor therapy of melanoma cells in C57Bl/6 (B16F1 50% survival p = 0.0026) and ENO1 241–260cit253 in HLA-DR4 transgenic mice (B16-DR4 50% survival p = 0.0048). In addition, ENO1 241–260cit253 induced therapy of pancreatic (Pan02-DR4 50% survival p = 0.0076) and lung (LLC/2-DR4 40% survival p = 0.0142) tumors in HLA-DR4 transgenic mice. The unmodified epitope induced no anti-tumor response. Minimal regression of class II negative B16 or LLC/2 tumor was seen, confirming direct recognition of MHC-II was required. Most tumors only express MHC-II in the presence of IFNγ; an IFNγ inducible model showed strong responses, with rejection of tumors in up to 90% of animals (p = 0.0001). In humans, a repertoire to ENO1 241–260cit253 was observed in healthy donors. This response was CD4 mediated and seen in people with a variety of HLA types suggesting a broad application for this vaccine in human cancer therapy.

Abstract A035: Citrullinated α-enolase as a novel target for cancer immunotherapy

Stressful conditions in the tumor microenvironment induce autophagy in cancer cells to promote their survival, however, autophagy also causes post-translational modification of proteins which are recognized by the immune system. In particular, modified self-antigens can trigger CD4+ T cell responses that can be exploited to boost antitumor immune defenses. We have previously investigated the ability of CD4 cells to target tumor-specific self-antigens modified by citrullination, which converts arginine residues in proteins to citrulline. These studies showed that vimentin, which is frequently expressed in cells during epithelial-to-mesenchymal transition of metastasizing epithelial tumors, is citrullinated and is a good target for anti-tumor immunity (Ref. 1). Immunization with citrullinated vimentin peptides induced IFNγ- and granzyme B-secreting CD4 T cells in response to autophagic tumor targets. Remarkably, a single immunization with modified peptide, up to 14 days after tumor implant, resulted in long term survival in 60-90% of animals with no associated toxicity. This antitumor response was dependent on CD4 cells and not CD8+ T cells. Due to its ubiquitous expression and abundance in most cells, the glycolytic enzyme α-enolase is a protein that is often citrullinated and degraded during autophagy and may represent a further novel antitumor target. In this study we demonstrate that immunization of C57Bl, HLA-DR4 and HLA-DP4 transgenic mice with citrullinated enolase peptides induces strong Th1/cytotoxic CD4 responses that efficiently target tumor cells. The Th1 cell repertoire to citrullinated enolase is also detectable in healthy donors and cancer patients. Immunization of mice with citrullinated enolase peptides led to tumor therapy in HLA-DP4 mice with established B16-DP4 tumors (70% survival p = 0.0058) and in HLA-DR4 transgenic mice with established B16F1-DR4 melanoma (50% survival; p = 0.0048) or Pan02-DR4 pancreatic tumors (survival 50%; p = 0.0076). The response was partially mediated by CD4 cytotoxic T cells as tumor therapy was observed against the HLA-DR4-expressing lung tumor LLC2 (40% survival; p = 0.0142) but no survival advantage was witnessed against LLC2 tumors which do not express class II MHC. As MHC-II is not expressed by the majority of tumors unless induced by IFNγ we designed an HLA-DR4 construct under expression of an IFNγ inducible promoter. Immunization of HLA-DR4 mice with citrullinated enolase peptides led to tumor therapy against the established B16F1-IFNγ inducible DR4 melanoma (90% survival p>0.0001). These results suggest that, similar to citrullinated vimentin, citrullinated α-enolase is a promising novel target for human cancer immunotherapy. References1. Brentville VA, Metheringham RL, Gunn B, Symonds P, Daniels I, Gijon M, Cook K, Xue W, Durrant LG (2016). Citrullinated vimentin presented on MHC-II in tumor cells is a target for CD4+ T cell-mediated antitumor immunity. Cancer Research 2016 Feb 1;76(3):548-60. Citation Format: Katherine Cook, Ian Daniels, Victoria Brentville, Rachael Metheringham, Wei Xue, Peter Symonds, Tracy Pitt, Mohammed Gijon, Lindy G. Durrant. Citrullinated α-enolase as a novel target for cancer immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A035.

Abstract A015: Protein arginine deiminase enzymes which citrullinate epitopes for MHC II presentation are independent predictors of survival in colorectal cancer

Citrullination of proteins is associated with a number of autoimmune diseases. Protein Arginine Deiminases (PADs) are a family of Ca2+ dependent enzymes that under cellular stress post-translationally convert arginine to citrulline within protein substrates to generate self-modified neo-antigens. It has been shown that presentation of citrullinated peptides on MHC class II stimulates CD4 T cells to mediate potent anti-tumor immunity (1). In this study we focus on the role of the PADI2 and PADI4 family members in colorectal cancer. Using a tissue microarray of colorectal cancers and compiling a comprehensive database of clinicopathological variables, the expression of PADI2 and PADI4 was assessed by immunohistochemistry in a study cohort. This data was used to correlate PADI2 and PADI4 expression with patient survival. In the study cohort 462 colorectal tumors were stained for PADI2 and PADI4. Of these 171 (37%) and 231 (50%) could not be evaluated for PADI2 and PADI4 staining respectively due to the absence of enough tissue core or no evaluable tumor cells (i.e. all stroma) in the core. Of the 291 evaluable colorectal tumors stained with a PADI2 specific antibody, only 18/291 (6.2%) tumors failed to stain. A further 153/291 (52.5%) stained weakly, 102/291 (35.1%) moderate and 18/291 (6.2%) stained strongly. Of the 231 evaluable colorectal tumors stained with a PADI4 specific antibody, no tumors failed to stain. All cases stained strongly for PADI4 expression within the nucleus. In the cytoplasm 63/231 (27.3%) stained weakly, 143/231 (61.9%) moderate and 25/231 (10.8%) stained strongly. PADI2 expression did not correlate with any clinicopathological variables whereas nuclear but not cytoplasmic PADI4 showed a strong association with histological type (p = 0.008). Kaplan-Meier analysis showed there was a correlation of PADI2 and cytoplasmic PADI4 expression with improved survival. Expression of PADI2 gave an increase in survival time from 44.8 months (95% CI 24.3 to 65.4) to 76.2 months (95% CI 69.9 to 82.4, log rank test, p = 0.012). Expression of cytoplasmic PADI4 increased survival time from 57.9 months (95% CI 43.6 to 72.3) to 77.3 months (95% CI 69.6 to 85.1, log rank test, p = 0.012). No significant correlation was observed between PADI2 and the cytoskeletal protein Vimentin or the glycolytic enzyme α-enolase both reported to be citrullinated by PAD enzymes. PADI2 expression was significantly associated with expression of the Nuclear antigen Ki67 (p = 0.046) a cellular marker for proliferation. Nuclear PADI4 significantly correlated with the cytoplasmic glycolytic enzyme α-enolase only (p = 0.001) and cytoplasmic PADI4 was highly significantly associated with α-enolase located in both the cytoplasm (p References 1. Brentville VA, Metheringham RL, Gunn B, Symonds P, Daniels I, Gijon M, Cook K, Xue W, Durrant LG (2016). Citrullinated vimentin presented on MHC-II in tumor cells is a target for CD4+ T cell-mediated antitumor immunity. Cancer Research 2016 Feb 1;76(3):548-60 Citation Format: R. Metheringham, M. Gijon, I. Daniels, K. Cook, P. Symonds, T. Pitt, W. Xue, V. Brentville, L. Durrant. Protein arginine deiminase enzymes which citrullinate epitopes for MHC II presentation are independent predictors of survival in colorectal cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A015.