Acidosis significantly alters immune checkpoint expression profiles of T cells from oesophageal adenocarcinoma patients.

Tumour acidosis contributes to cancer progression by inhibiting anti-tumour immunity. However, the effect of acidosis on anti-tumour T cell phenotypes in oesophageal adenocarcinoma (OAC) is unknown. Therefore, this study investigated the effect of acidosis on anti-tumour T cell profiles and if immune checkpoint blockade (ICB) could enhance anti-tumour T cell immunity under acidosis. Acidic conditions substantially altered immune checkpoint expression profiles of OAC patient-derived T cells, upregulating TIM-3, LAG-3 and CTLA-4. Severe acidosis (pH 5.5) significantly decreased the percentage of central memory CD4+ T cells, an effect that was attenuated by ICB treatment. ICB increased T cell production of IFN-γ under moderate acidosis (pH 6.6) but not severe acidosis (pH 5.5) and decreased IL-10 production by T cells under severe acidic conditions only. A link between lactate and metastasis was also depicted; patients with nodal metastasis had higher serum lactate levels (p = 0.07) which also positively correlated with circulating levels of pro-angiogenic factor Tie-2. Our findings establish that acidosis-induced upregulation of immune checkpoints on T cells may potentially contribute to immune evasion and disease progression in OAC. However, acidic conditions curtailed ICB efficacy, supporting a rationale for utilizing systemic oral buffers to neutralize tumour acidity to improve ICB efficacy. Study schematic-PBMCs were isolated from OAC patients (A) and expanded ex vivo for 7 days using anti-CD3/28 +IL-2 T cell activation protocol (B) and further cultured for 48 h under increasing acidic conditions in the absence or presence of immune checkpoint blockade (nivolumab, ipilimumab or dual nivolumab + ipilimumab) (C). Immunophenotyping was then carried out to assess immune checkpoint expression profiles and anti-tumour T cell phenotypes (D). Serum lactate was assessed in OAC patients (E-F) and levels were correlated with patient demographics (G) and the levels of circulating immune/pro-angiogenic cytokines that were determined by multiplex ELISA (H). Key Findings-severe acidic conditions upregulated multiple immune checkpoints on T cells (I). Efficacy of ICB was curtailed under severe acidic conditions (J). Circulating lactate levels positively correlated with circulating levels of pro-angiogenic factor tie-2 and higher serum lactate levels were found in patients who had nodal metastasis (K).

The tumour immune microenvironment in oesophageal cancer.

Oesophageal cancer (OC) is an inflammation-associated malignancy linked to gastro-oesophageal reflux disease, obesity and tobacco use. Knowledge of the microenvironment of oesophageal tumours is relevant to our understanding of the development of OC and its biology, and has major implications for understanding the response to standard therapies and immunotherapies, as well as for uncovering novel targets. In this context, we discuss what is known about the TME in OC from tumour initiation to development and progression, and how this is relevant to therapy sensitivity and resistance in the two major types of OC. We provide an immunological characterisation of the OC TME and discuss its prognostic implications with specific comparison with the Immunoscore and immune-hot, -cold, altered-immunosuppressed and -altered-excluded models. Targeted therapeutics for the TME under pre-clinical and clinical investigation in OCs are also summarised. A deeper understanding of the TME will enable the development of combination approaches to concurrently target the tumour cells and TME delivering precision medicine to OC patients.

Unconventional T cells - New players in antifungal immunity.

Life-threatening invasive fungal diseases (IFD) are increasing in incidence, especially in immunocompromised patients and successful resolution of IFD requires a variety of different immune cells. With the limited repertoire of available antifungal drugs there is a need for more effective therapeutic strategies. This review interrogates the evidence on the human immune response to the main pathogens driving IFD, with a focus on the role of unconventional lymphocytes e.g. natural killer (NK) cells, gamma/delta (γδ) T cells, mucosal associated invariant T (MAIT) cells, invariant natural killer T (iNKT) cells and innate lymphoid cells (ILC). Recent discoveries and new insights into the roles of these novel lymphocyte groups in antifungal immunity will be discussed, and we will explore how an improved understanding of antifungal action by lymphocytes can inform efforts to improve antifungal treatment options.

Tissue distribution of γδ T cell subsets in oesophageal adenocarcinoma.

The global obesity epidemic is contributing to increased prevalence of diseases fuelled by chronic inflammation, including cancer. Oesophageal adenocarcinoma (OAC) is an obesity-associated malignancy with increasing prevalence, dismal prognosis, and severely dysregulated immune processes. We previously reported that αß T cells migrate to omentum and liver in OAC and contribute to inflammation in these tissues. Here, we assessed the tissue distribution and phenotype of gamma/delta (γδ) T cells in the blood, omentum, liver and tumour of OAC patients. Our data show that the Vδ1 and Vδ3 subsets of γδ T cells are most prevalent in omentum and liver of OAC patients. Furthermore, γδ T cells are predominantly pro-inflammatory in these tissues, and co-express IFN-γ and IL-17. Moreover, γδ T cells exhibit cytotoxic capabilities in OAC omentum and liver. This study provides the first indication that γδ T cells contribute to obesity-associated inflammation in OAC and might be exploited therapeutically.

Interleukin-17 producing mucosal associated invariant T cells - emerging players in chronic inflammatory diseases?

Mucosal associated invariant T (MAIT) cells are a population of evolutionarily conserved T cells, which express an invariant T cell receptor (TCR) and represent a significant subset of innate-like T cells in humans, yet their role in immunity is still emerging. Unlike conventional αß T cells, MAIT cells are not restricted by MHC molecules, but instead uniquely recognize microbially derived vitamin metabolites presented by the MHC-I like molecule MR1. MAIT cells are enriched in mucosal sites and tissues including liver and adipose tissue where they are thought to play an important role in immunosurveillance and immunity against microbial infection. In addition to their putative role in antimicrobial immunity, recent research on MAIT cells, in particular IL-17 producing MAIT cells, has demonstrated their involvement in numerous chronic inflammatory conditions. In this review, we give an overview of the work to date on the function and subsets of MAIT cells. We also examine the role of IL-17 producing MAIT cells in chronic inflammatory diseases ranging from autoimmune conditions, metabolic diseases to cancer. Furthermore, we discuss the most recent findings from the clinic that might help deepen our understanding about the biology of MAIT cells.

Characterising the prognostic potential of HLA-DR during colorectal cancer development.

HLA-DR, an MHC class II molecule that mediates antigen presentation, is a favourable prognostic indicator in colorectal cancer (CRC). However, the dynamics and location of HLA-DR expression during CRC development are unclear. We aimed to define HLA-DR expression by immunohistochemistry in colorectal epithelium and stromal tissue at different stages of cancer development, assessing non-neoplastic colorectal adenocarcinoma-adjacent tissue, adenomas and carcinoma tissues, and to associate HLA-DR levels with clinical outcomes. Patients with higher than median HLA-DR expression survived at least twice as long as patients with lower expression. This association was significant for HLA-DR staining in the colorectal carcinoma epithelium (n = 152, p = 0.011, HR 1.9, 95% CI 1.15-3.15) and adjacent non-neoplastic epithelium (n = 152, p < 0.001, HR 2.7, 95% CI 1.59-4.66), but not stroma. In stage II cases, however, the prognostic value of HLA-DR expression was significant only in adjacent non-neoplastic tissues, for both epithelium (n = 63, p = 0.015, HR 3.6, 95% CI 1.279-10.25) and stroma (n = 63, p = 0.018, HR 5.07, 95% CI 1.32-19.49). HLA-DR was lower in carcinoma tissue compared to matched adenomas (n = 35), in epithelium (p < 0.01) and stroma (p < 0.001). HLA-DR was further reduced in late-stage carcinoma (n = 101) compared to early stage (n = 105), in epithelium (p < 0.001) and stroma (p < 0.01). HLA-DR expression was lower (p < 0.05) in the adjacent non-neoplastic epithelium of patients with cancer recurrence. We demonstrate a progressive loss of HLA-DR in epithelial and stromal tissue compartments during CRC development and show prognostic ability in carcinoma-adjacent non-neoplastic tissues, highlighting the importance of this molecule in the anti-cancer immune response. These findings may have wider implications for immunotherapeutic interventions.

Combining 1,4-dihydroxy quininib with Bevacizumab/FOLFOX alters angiogenic and inflammatory secretions in ex vivo colorectal tumors.

BACKGROUND: Colorectal cancer (CRC) is the second most common cause of cancer-related mortality worldwide with one in every five patients diagnosed with metastatic CRC (mCRC). In mCRC cases, the 5-year survival rate remains at approximately 14%, reflecting the lack of effectiveness of currently available treatments such as the anti-VEGF targeting antibody Bevacizumab combined with the chemotherapy folinic acid, fluorouracil and oxaliplatin (FOLFOX). Approximately 60% of patients do not respond to this combined treatment. Furthermore, Bevacizumab inhibits dendritic cell (DC) maturation in poor responders, a key process for tumor eradication. METHOD: Following drug treatment, secreted expression levels of angiogenic and inflammatory markers in tumor conditioned media generated from human ex vivo colorectal tumors were measured by ELISA. Dendritic cell phenotypic and maturation markers were assessed by flow cytometry. RESULTS: Our novel compound, 1,4-dihydroxy quininib, acts in an alternative pathway compared to the approved therapy Bevacizumab. 1,4-dihydroxy quininib alone, and in combination with Bevacizumab or FOLFOX significantly reduced TIE-2 expression which is involved in the promotion of tumor vascularization. Combination treatment with 1,4-dihydroxy quininib significantly increased the expression level of DC phenotypic and maturation markers. CONCLUSION: Our results indicate the anti-angiogenic small molecule 1,4-dihydroxy quininib could be an alternative novel treatment in combination therapy for CRC patients.

The tumour microenvironment of the upper and lower gastrointestinal tract differentially influences dendritic cell maturation.

BACKGROUND: Only 10-30% of oesophageal and rectal adenocarcinoma patients treated with neoadjuvant chemoradiotherapy have a complete pathological response. Inflammatory and angiogenic mediators in the tumour microenvironment (TME) may enable evasion of anti-tumour immune responses. METHODS: The TME influence on infiltrating dendritic cells (DCs) was modelled by treating immature monocyte-derived DCs with Tumour Conditioned Media (TCM) from distinct gastrointestinal sites, prior to LPS-induced maturation. RESULTS: Cell line conditioned media from gastrointestinal cell lines inhibited LPS-induced DC markers and TNF-α secretion. TCM generated from human tumour biopsies from oesophageal, rectal and colonic adenocarcinoma induced different effects on LPS-induced DC markers - CD54, CD80, HLA-DR, CD86 and CD83 were enhanced by oesophageal cancer; CD80, CD86 and CD83 were enhanced by rectal cancer, whereas CD54, HLA-DR, CD86, CD83 and PD-L1 were inhibited by colonic cancer. Notably, TCM from all GI cancer types inhibited TNF-α secretion. Additionally, TCM from irradiated biopsies inhibited DC markers. Profiling the TCM showed that IL-2 levels positively correlated with maturation marker CD54, while Ang-2 and bFGF levels negatively correlated with CD54. CONCLUSION: This study identifies that there are differences in DC maturational capacity induced by the TME of distinct gastrointestinal cancers. This could potentially have implications for anti-tumour immunity and response to radiotherapy.

HLA-DR expression in tumor epithelium is an independent prognostic indicator in esophageal adenocarcinoma patients.

Esophageal adenocarcinoma (EAC) is an aggressive cancer with poor prognosis, and incidence is increasing rapidly in the Western world. Measurement of immune markers has been shown to have prognostic significance in a growing number of cancers, but whether this is true for EAC has yet to be evaluated. This study aimed to characterize HLA-DR expression in the esophagus across the inflammation to cancer progression sequence and to assess the prognostic significance of HLA-DR expression in EAC. Tissue microarrays (TMA) were constructed from esophageal tissue taken from patients at different stages in the cancer progression sequence; normal, esophagitis, Barrett's esophagus (BE), low- and high-grade dysplasia (LGD, HGD) and EAC. HLA-DR expression in tissue epithelium and stroma was assessed by immunohistochemistry. HLA-DR expression increased early in the inflammation to cancer progression sequence; with higher expression detected in esophagitis and BE compared to normal tissue. Patients with low (<50%) HLA-DR expression in the EAC tumor epithelium had significantly worse survival outcomes, compared to those with high expression, in both the tumor core (hazard ratio, HR = 2.178, p = 0.024, n = 70) and leading edge (HR = 2.86, p = 0.013, n = 41). Multivariate analysis demonstrated that low HLA-DR expression in leading edge tumor epithelium was an independent predictor of poor survival, associated with a 2.8-fold increase in disease-associated death (p = 0.023). This study shows that HLA-DR is an independent prognostic marker in EAC tumor epithelium. This may have implications for patient stratification strategies as well as EAC tumor immunology.

Effects of a multimodal rehabilitation programme on inflammation and oxidative stress in oesophageal cancer survivors: the ReStOre feasibility study.

PURPOSE: Physical, nutritional and quality-of-life compromises are known sequelae of oesophageal cancer (OC) treatment. Inflammation and oxidative stress may be relevant to adverse consequences. Multimodal rehabilitation involving exercise and diet prescription may attenuate some of the negative consequences and optimise survivorship, and this was assessed in this feasibility study in OC patients at least 1 year post-oesophagectomy. METHODS: The 12-week programme included supervised and home-based exercise, dietetic counselling to ensure energy balance and multidisciplinary education. Baseline and post-intervention assessments examined aerobic fitness, physical activity and body composition. Serum interleukin (IL)-1ß, tumour necrosis factor (TNF)-α, IL-6 and IL-8 were measured via multiplex arrays. Lactate secretion, lipid peroxidation (4-HNE) and oxidative stress (8-iso-PGF2α) were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Twelve patients (mean (SD) age 64(1.29) years) participated. IL-8 reduced significantly from pre- to post-intervention (percentage change -11.25 % (95 % CI -20.98 to -1.51 %), p = 0.03), and there was a non-significant trend towards lower expression patterns of other inflammatory mediators. At baseline, inflammatory status correlated inversely with sedentary behaviour (IL-6 rho = -0.74, IL-8 rho = -0.59, TNF-α rho = -0.69; p < 0.05). While energy metabolism did not change, post-intervention lactate concentration correlated strongly and inversely with aerobic fitness (rho = -0.68, p = 0.02). Body composition was maintained throughout the intervention. CONCLUSIONS: Results suggest that multimodal rehabilitation following OC treatment reduced inflammatory status without compromising body composition. Findings will be further examined in a larger randomised controlled trial.

Cutting edge: CD1d restriction and Th1/Th2/Th17 cytokine secretion by human Vδ3 T cells.

Human γδ T cells expressing the Vδ3 TCR make up a minor lymphocyte subset in blood but are enriched in liver and in patients with some chronic viral infections and leukemias. We analyzed the frequencies, phenotypes, restriction elements, and functions of fresh and expanded peripheral blood Vδ3 T cells. Vδ3 T cells accounted for ~0.2% of circulating T cells, included CD4(+), CD8(+), and CD4(-)CD8(-) subsets, and variably expressed CD56, CD161, HLA-DR, and NKG2D but neither NKG2A nor NKG2C. Vδ3 T cells were sorted and expanded by mitogen stimulation in the presence of IL-2. Expanded Vδ3 T cells recognized CD1d but not CD1a, CD1b, or CD1c. Upon activation, they killed CD1d(+) target cells, released Th1, Th2, and Th17 cytokines, and induced maturation of dendritic cells into APCs. Thus, Vδ3 T cells are glycolipid-reactive T cells with distinct Ag specificities but functional similarities to NKT cells.

Energy Metabolism, Metabolite, and Inflammatory Profiles in Human Ex Vivo Adipose Tissue Are Influenced by Obesity Status, Metabolic Dysfunction, and Treatment Regimes in Patients with Oesophageal Adenocarcinoma.

Oesophageal adenocarcinoma (OAC) is a poor prognosis cancer with limited response rates to current treatment modalities and has a strong link to obesity. To better elucidate the role of visceral adiposity in this disease state, a full metabolic profile combined with analysis of secreted pro-inflammatory cytokines, metabolites, and lipid profiles were assessed in human ex vivo adipose tissue explants from obese and non-obese OAC patients. These data were then related to extensive clinical data including obesity status, metabolic dysfunction, previous treatment exposure, and tumour regression grades. Real-time energy metabolism profiles were assessed using the seahorse technology. Adipose explant conditioned media was screened using multiplex ELISA to assess secreted levels of 54 pro-inflammatory mediators. Targeted secreted metabolite and lipid profiles were analysed using Ultra-High-Performance Liquid Chromatography coupled with Mass Spectrometry. Adipose tissue explants and matched clinical data were collected from OAC patients (n = 32). Compared to visceral fat from non-obese patients (n = 16), visceral fat explants from obese OAC patients (n = 16) had significantly elevated oxidative phosphorylation metabolism profiles and an increase in Eotaxin-3, IL-17A, IL-17D, IL-3, MCP-1, and MDC and altered secretions of glutamine associated metabolites. Adipose explants from patients with metabolic dysfunction correlated with increased oxidative phosphorylation metabolism, and increases in IL-5, IL-7, SAA, VEGF-C, triacylglycerides, and metabolites compared with metabolically healthy patients. Adipose explants generated from patients who had previously received neo-adjuvant chemotherapy (n = 14) showed elevated secretions of pro-inflammatory mediators, IL-12p40, IL-1α, IL-22, and TNF-ß and a decreased expression of triacylglycerides. Furthermore, decreased secreted levels of triacylglycerides were also observed in the adipose secretome of patients who received the chemotherapy-only regimen FLOT compared with patients who received no neo-adjuvant treatment or chemo-radiotherapy regimen CROSS. For those patients who showed the poorest response to currently available treatments, their adipose tissue was associated with higher glycolytic metabolism compared to patients who had good treatment responses. This study demonstrates that the adipose secretome in OAC patients is enriched with mediators that could prime the tumour microenvironment to aid tumour progression and attenuate responses to conventional cancer treatments, an effect which appears to be augmented by obesity and metabolic dysfunction and exposure to different treatment regimes.

The role of unconventional T cells in COVID-19.

COVID-19 is a respiratory disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It was first documented in late 2019, but within months, a worldwide pandemic was declared due to the easily transmissible nature of the virus. Research to date on the immune response to SARS-CoV-2 has focused largely on conventional B and T lymphocytes. This review examines the emerging role of unconventional T cell subsets, including γδ T cells, invariant natural killer T (iNKT) cells and mucosal associated invariant T (MAIT) cells in human SARS-CoV-2 infection.Some of these T cell subsets have been shown to play protective roles in anti-viral immunity by suppressing viral replication and opsonising virions of SARS-CoV. Here, we explore whether unconventional T cells play a protective role in SARS-CoV-2 infection as well. Unconventional T cells are already under investigation as cell-based immunotherapies for cancer. We discuss the potential use of these cells as therapeutic agents in the COVID-19 setting. Due to the rapidly evolving situation presented by COVID-19, there is an urgent need to understand the pathogenesis of this disease and the mechanisms underlying its immune response. Through this, we may be able to better help those with severe cases and lower the mortality rate by devising more effective vaccines and novel treatment strategies.

SARS-CoV-2 spike and nucleocapsid proteins fail to activate human dendritic cells or γδ T cells.

γδ T cells are thought to contribute to immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the mechanisms by which they are activated by the virus are unknown. Using flow cytometry, we investigated if the two most abundant viral structural proteins, spike and nucleocapsid, can activate human γδ T cell subsets, directly or in the presence of dendritic cells (DC). Both proteins failed to induce interferon-γ production by Vδ1 or Vδ2 T cells within fresh mononuclear cells or lines of expanded γδ T cells generated from healthy donors, but the same proteins stimulated CD3+ cells from COVID-19 patients. The nucleocapsid protein stimulated interleukin-12 production by DC and downstream interferon-γ production by co-cultured Vδ1 and Vδ2 T cells, but protease digestion and use of an alternative nucleocapsid preparation indicated that this activity was due to contaminating non-protein material. Thus, SARS-CoV-2 spike and nucleocapsid proteins do not have stimulatory activity for DC or γδ T cells. We propose that γδ T cell activation in COVID-19 patients is mediated by immune recognition of viral RNA or other structural proteins by γδ T cells, or by other immune cells, such as DC, that produce γδ T cell-stimulatory ligands or cytokines.

Differential Effects of Dietary versus Exercise Intervention on Intrahepatic MAIT Cells and Histological Features of NAFLD.

BACKGROUND: Mucosal-associated invariant T (MAIT) cells promote inflammation in obesity and are implicated in the progression of non-alcoholic fatty liver disease (NAFLD). However, as the intrahepatic MAIT cell response to lifestyle intervention in NAFLD has not been investigated, this work aimed to examine circulating and intrahepatic MAIT cell populations in patients with NAFLD, after either 12 weeks of dietary intervention (DI) or aerobic exercise intervention (EI). METHODS: Multicolour flow cytometry was used to immunophenotype circulating and intrahepatic MAIT cells and measure MAIT cell expression (median fluorescence intensity, MFI) of the activation marker CD69 and apoptotic marker CD95. Liver histology, clinical parameters, and MAIT cell populations were assessed at baseline (T0) and following completion (T1) of DI or EI. RESULTS: Forty-five patients completed the study. DI participants showed decreased median (interquartile range) expression of the activation marker CD69 on circulating MAIT cells (T0: 104 (134) versus T1 27 (114) MFI; p = 0.0353) and improvements in histological steatosis grade post-intervention. EI participants showed increased expression of the apoptotic marker CD95, both in circulating (T0: 1549 (888) versus T1: 2563 (1371) MFI; p = 0.0043) and intrahepatic MAIT cells (T0: 2724 (862) versus T1: 3117 (1622) MFI; p = 0.0269). Moreover, the percentage of intrahepatic MAIT cells significantly decreased after EI (T0: 11.1 (14.4) versus T1: 5.3 (9.3)%; p = 0.0029), in conjunction with significant improvements in fibrosis stage and hepatocyte ballooning. CONCLUSIONS: These data demonstrate independent benefits from dietary and exercise intervention and suggest a role for intrahepatic MAIT cells in the observed histological improvements in NAFLD.

Impact of radiotherapy on the immune landscape in oesophageal adenocarcinoma.

BACKGROUND: In the contemporary era of cancer immunotherapy, an abundance of clinical and translational studies have reported radiotherapy (RT) and immunotherapies as a viable option for immunomodulation of many cancer subtypes, with many related clinical trials ongoing. In locally advanced disease, chemotherapy or chemoradiotherapy followed by surgical excision of the tumour remain the principal treatment strategy in oesophageal adenocarcinoma (OAC), however, the use of the host immune system to improve anti-tumour immunity is rapidly garnering increased support in the curative setting. AIM: To immunophenotype OAC patients' immune checkpoint (IC) expression with and without radiation and evaluate the effects of checkpoint blockade on cell viability. METHODS: In the contemporary era of cancer immunotherapy, an abundance of studies have demonstrated that combination RT and IC inhibitors (ICIs) are effective in the immunomodulation of many cancer subtypes, with many related clinical trials ongoing. Although surgical excision and elimination of tumour cells by chemotherapy or chemoradiotherapy remains the gold standard approach in OAC, the propagation of anti-tumour immune responses is rapidly garnering increased support in the curative setting. The aim of this body of work was to immunophenotype OAC patients' IC expression with and without radiation and to establish the impact of checkpoint blockade on cell viability. This study was a hybrid combination of in vitro and ex vivo models. Quantification of serum immune proteins was performed by enzyme-linked immunosorbent assay. Flow cytometry staining was performed to evaluate IC expression for in vitro OAC cell lines and ex vivo OAC biopsies. Cell viability in the presence of radiation with and without IC blockade was assessed by a cell counting kit-8 assay. RESULTS: We identified that conventional dosing and hypofractionated approaches resulted in increased IC expression (PD-1, PD-L1, TIM3, TIGIT) in vitro and ex vivo in OAC. There were two distinct subcohorts with one demonstrating significant upregulation of ICs and the contrary in the other cohort. Increasing IC expression post RT was associated with a more aggressive tumour phenotype and adverse features of tumour biology. The use of anti-PD-1 and anti-PD-L1 immunotherapies in combination with radiation resulted in a significant and synergistic reduction in viability of both radiosensitive and radioresistant OAC cells in vitro. Interleukin-21 (IL-21) and IL-31 significantly increased, with a concomitant reduction in IL-23 as a consequence of 4 Gray radiation. Similarly, radiation induced an anti-angiogenic tumour milieu with reduced expression of vascular endothelial growth factor-A, basic fibroblast growth factor, Flt-1 and placental growth factor. CONCLUSION: The findings of the current study demonstrate synergistic potential for the use of ICIs and ionising radiation to potentiate established anti-tumour responses in the neoadjuvant setting and is of particular interest in those with advanced disease, adverse features of tumour biology and poor treatment responses to conventional therapies.

The Impact of Esophageal Oncological Surgery on Perioperative Immune Function; Implications for Adjuvant Immune Checkpoint Inhibition.

Background: Immune checkpoint inhibitors (ICIs) are being investigated for their role as an adjunct in the multimodal treatment of esophageal adenocarcinoma (EAC). The most effective time to incorporate ICIs remains unknown. Our study profiles systemic anti-tumor immunity perioperatively to help inform the optimal timing of ICIs into current standards of care for EAC patients. Methods: Systemic immunity in 11 EAC patients was phenotyped immediately prior to esophagectomy (POD-0) and post-operatively (POD)-1, 3, 7 and week 6. Longitudinal serological profiling was conducted by ELISA. The frequency of circulating lymphocytes, activation status, immune checkpoint expression and damage-associated molecular patterns was assessed by flow cytometry. Results: The frequency of naïve T-cells significantly increased in circulation post-esophagectomy from POD-0 to POD-7 (p<0.01) with a significant decrease in effector memory T-cells by POD7 followed by a subsequent increase by week 6 (p<0.05). A significant increase in activated circulating CD27+ T-cells was observed from POD-0 to POD-7 (p<0.05). The percentage of PD-1+ and CTLA-4+ T-cells peaked on POD-1 and was significantly decreased by week 6 (p<0.01). There was a significant increase in soluble PD-1, PD-L2, TIGIT and LAG-3 from POD-3 to week 6 (p<0.01). Increased checkpoint expression correlated with those who developed metastatic disease early in their postoperative course. Th1 cytokines and co-stimulatory factors decreased significantly in the immediate post-operative setting, with a reduction in IFN-γ, IL-12p40, IL-1RA, CD28, CD40L and TNF-α. A simultaneous increase was observed in Th2 cytokines in the immediate post-operative setting, with a significant increase in IL-4, IL-10, IL-16 and MCP-1 before returning to preoperative levels at week 6. Conclusion: Our study highlights the prevailing Th2-like immunophenotype post-surgery. Therefore, shifting the balance in favour of a Th1-like phenotype would offer a potent therapeutic approach to promote cancer regression and prevent recurrence in the adjuvant setting and could potentially propagate anti-tumour immune responses perioperatively if administered in the immediate neoadjuvant setting. Consequently, this body of work paves the way for further studies and appropriate trial design is needed to further interrogate and validate the use of ICI in the multimodal treatment of locally advanced disease in the neoadjuvant and adjuvant setting.

Identification of plasma proteins associated with oesophageal cancer chemotherapeutic treatment outcomes using SWATH-MS.

Oesophageal adenocarcinoma (OAC) is an aggressive cancer with a five-year survival of <15%. Current chemotherapeutic strategies only benefit a minority (20-30%) of patients and there are no methods available to differentiate between responders and non-responders. We performed quantitative proteomics using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) on albumin/IgG-depleted and non-depleted plasma samples from 23 patients with locally advanced OAC prior to treatment. Individuals were grouped based on tumour regression (TRG) score (TRG1/2/3 vs TRG4/5) after chemotherapy, and differentially abundant proteins were compared. Protein depletion of highly abundant proteins led to the identification of around twice as many proteins. SWATH-MS revealed significant quantitative differences in the abundance of several proteins between the two groups. These included complement c1q subunit proteins, C1QA, C1QB and C1QC, which were of higher abundance in the low TRG group. Of those that were found to be of higher abundance in the high TRG group, glutathione S-transferase pi (GSTP1) exhibited the lowest p-value and highest classification accuracy and Cohen's kappa value. Concentrations of these proteins were further examined using ELISA-based assays. This study provides quantitative information relating to differences in the plasma proteome that underpin response to chemotherapeutic treatment in oesophageal cancers. SIGNIFICANCE: Oesophageal cancers, including oesophageal adenocarcinoma (OAC) and oesophageal gastric junction cancer (OGJ), are one of the leading causes of cancer mortality worldwide. Curative therapy consists of surgery, either alone or in combination with adjuvant or neoadjuvant chemotherapy or radiation, or combination chemoradiotherapy regimens. There are currently no clinico-pathological means of predicting which patients will benefit from chemotherapeutic treatments. There is therefore an urgent need to improve oesophageal cancer disease management and treatment strategies. This work compared proteomic differences in OAC patients who responded well to chemotherapy as compared to those who did not, using quantitative proteomics prior to treatment commencement. SWATH-MS analysis of plasma (with and without albumin/IgG-depletion) from OAC patients prior to chemotherapy was performed. This approach was adopted to determine whether depletion offered a significant improvement in peptide coverage. Resultant datasets demonstrated that depletion increased peptide coverage significantly. Additionally, there was good quantitative agreement between commonly observed peptides. Data analysis was performed by adopting both univariate as well as multivariate analysis strategies. Differentially abundant proteins were identified between treatment response groups based on tumour regression grade. Such proteins included complement C1q sub-components and GSTP1. This study provides a platform for further work, utilising larger sample sets across different treatment regimens for oesophageal cancer, that will aid the development of 'treatment response prediction assays' for stratification of OAC patients prior to chemotherapy.

Activation of human invariant natural killer T cells with a thioglycoside analogue of α-galactosylceramide.

Activation of CD1d-restricted invariant NKT (iNKT) cells with the glycolipid α-galactosylceramide (α-GalCer) confers protection against disease in murine models, however, clinical trials in humans have had limited impact. We synthesized a novel thioglycoside analogue of α-GalCer, denoted α-S-GalCer, and tested its efficacy for stimulating human iNKT cells in vitro. α-S-GalCer stimulated cytokine release by iNKT cells in a CD1d-dependent manner and primed CD1d(+) target cells for lysis. α-S-GalCer-stimulated iNKT cells induced maturation of monocyte-derived dendritic cells into antigen-presenting cells that released IL-12 and small amounts of IL-10. The nature and potency of α-S-GalCer and α-GalCer in human iNKT cell activation were similar. However, in contrast to α-GalCer, α-S-GalCer did not activate murine iNKT cells in vivo. Because of its enhanced stability in biological systems, α-S-GalCer may be superior to α-GalCer as a parent compound for developing adjuvant therapies for humans.

Examination and characterisation of the effect of amitriptyline therapy for chronic neuropathic pain on neuropeptide and proteomic constituents of human cerebrospinal fluid.

INTRODUCTION: Amitriptyline is prescribed to reduce the intensity of chronic neuropathic pain. There is a paucity of validated in vivo evidence in humans regarding amitriptyline's mechanism of action. We examined the effect of amitriptyline therapy on cerebrospinal fluid (CSF) neuropeptides and proteome in patients with chronic neuropathic pain to identify potential mechanisms of action of amitriptyline. METHODS: Patients with lumbar radicular neuropathic pain were selected for inclusion with clinical and radiological signs and a >50% reduction in pain in response to a selective nerve root block. Baseline (pre-treatment) and 8-week (post-treatment) pain scores with demographics were recorded. CSF samples were taken at baseline (pre-treatment) and 8 weeks after amitriptyline treatment (post-treatment). Proteome analysis was performed using mass spectrometry and secreted cytokines, chemokines and neurotrophins were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: A total of 9/16 patients experienced a >30% reduction in pain after treatment with amitriptyline and GO analysis demonstrated that the greatest modulatory effect was on immune system processes. KEGG analysis also identified a reduction in PI3K-Akt and MAPK signalling pathways in responders but not in non-responders. There was also a significant decrease in the chemokine eotaxin-1 (p â€‹= â€‹0.02) and a significant increase in the neurotrophin VEGF-A (p â€‹= â€‹0.04) in responders. CONCLUSION: The CSF secretome and proteome was modulated in responders to amitriptyline verifying many pre-clinical and in vitro models. The predominant features were immunomodulation with a reduction in pro-inflammatory pathways of neuronal-glia communications and evidence of a neurotrophic effect.