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Aim: Use of immune checkpoint blockade to enhance T cell-mediated immunity within the hostile tumour microenvironment (TME) is an attractive approach in oesophageal adenocarcinoma (OAC). This study explored the effects of the hostile TME, including nutrient deprivation and hypoxia, on immune checkpoint (IC) expression and T cell phenotypes, and the potential use of nivolumab to enhance T cell function under such conditions., Methods and Results: ICs were upregulated on stromal immune cells within the tumour including PD-L2, CTLA-4 and TIGIT. OAC patient-derived PBMCs co-cultured with OE33 OAC cells upregulated LAG-3 and downregulated the co-stimulatory marker CD27 on T cells, highlighting the direct immunosuppressive effects of tumour cells on T cells. Hypoxia and nutrient deprivation altered the secretome of OAC patient-derived PBMCs, which induced upregulation of PD-L1 and PD-L2 on OE33 OAC cells thus enhancing an immune-resistant phenotype. Importantly, culturing OAC patient-derived PBMCs under dual hypoxia and glucose deprivation, reflective of the conditions within the hostile TME, upregulated an array of ICs on the surface of T cells including PD-1, CTLA-4, A2aR, PD-L1 and PD-L2 and decreased expression of IFN-γ by T cells. Addition of nivolumab under these hostile conditions decreased the production of pro-tumorigenic cytokine IL-10., Conclusion: Collectively, these findings highlight the immunosuppressive crosstalk between tumour cells and T cells within the OAC TME. The ability of nivolumab to suppress pro-tumorigenic T cell phenotypes within the hostile TME supports a rationale for the use of immune checkpoint blockade to promote anti-tumour immunity in OAC. Study schematic: (A) IC expression profiles were assessed on CD45 + cells in peripheral whole blood and infiltrating tumour tissue from OAC patients in the treatment-naïve setting. (B) PBMCs were isolated from OAC patients and expanded ex vivo for 5 days using anti-CD3/28 + IL-2 T cell activation protocol and then co-cultured for 48 h with OE33 cells. T cell phenotypes were then assessed by flow cytometry. (C) PBMCs were isolated from OAC patients and expanded ex vivo for 5 days using anti-CD3/28 + IL-2 T cell activation protocol and then further cultured under conditions of nutrient deprivation or hypoxia for 48 h and T cell phenotypes were then assessed by flow cytometry., Key Findings: (A) TIGIT, CTLA-4 and PD-L2 were upregulated on CD45 + immune cells and CTLA-4 expression on CD45 + cells correlated with a subsequent decreased response to neoadjuvant regimen. (B) Following a 48 h co-culture with OE33 cells, T cells upregulated LAG-3 and decreased CD27 co-stimulatory marker. (C) Nutrient deprivation and hypoxia upregulated a range of ICs on T cells and decreased IFN-γ production by T cells. Nivolumab decreased IL-10 production by T cells under nutrient deprivation-hypoxic conditions., (© 2022. The Author(s).)

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)., (© 2022. The Author(s).)

Introduction: Chronic radicular neuropathic pain is a major clinical problem with a life time prevalence of more than 50%. Pulsed radiofrequency (PRF) treatment is a recognised therapy. However, the pathophysiology of chronic neuropathic pain (CNP) and the mechanism of action of PRF remains ill-defined. Improving our knowledge of the mechanisms of CNP and PRF action will enhance our ability to treat patients with this common debilitating problem more effectively. This study aims to characterise the CSF cellular and peptide constituents in patients with CNP and the effect of pulsed radiofrequency (PRF) on these constituents and reported pain., Materials and Methods: Prospective randomised tripled-blinded control trial of patients receiving PRF treatment versus sham for radicular pain. All patients received local anaesthetic to the appropriate dermatome to confirm diagnosis. Clinical assessment using standard clinical assessment tools and examination of CSF using flow cytometry and ELISA for cellular and peptide constituents was carried out before and 3 months after treatment., Results: Ten patients were randomised to PRF (n = 5) or Sham (n = 5) treatment. PRF resulted in a significant reduction in pain score (NRS) at 3 months (6.8 to 2.6, p < .05). PRF reduced the TNF-α concentration and CD3+ count in CSF. CD4/CD8 ratio of patients with CNP was lower than historical controls (1.4 versus 3.0-4.2). The majority of CD3+ cells in the CNP patients were activated effector memory cells (80%) versus the surveillance central memory cells (85%) seen in healthy controls., Conclusions: PRF is superior to local anaesthetic administration for the management of radicular pain and is associated with CSF constituent modulation in vivo. Patients with CNP have lymphocyte characteristics which suggest immune activation., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020. Published by Elsevier B.V.)

Amitriptyline is prescribed for treating the symptoms of neuroinflammatory disorders including neuropathic pain and fibromyalgia. As amitriptyline has evidence of modulating the neuroimmune interface; the effects of amitriptyline treatment on T-cell phenotype and function were examined in vitro. Peripheral blood mononuclear cells(PBMCs) were isolated and treated with amitriptyline, nortriptyline and a combination of both drugs. Toxicity for T-cells was assessed by Annexin V/Propidium Iodide staining. Activation status and cytokine expression by T-cells post treatment was assessed by flow cytometry. The levels of secreted cytokines, chemokines and neurotrophins were measured by ELISA in the supernatants. There was no significant increase in T-cell death following 24 or 48 h compared to controls. There were significantly lower frequencies of CD8 + T-cells after treatment with amitriptyline, nortriptyline and a combination of both compared to a Vehicle Control(VC)(p<0.001). The frequencies of naive CD8 + CD45RA + cells were significantly lower after amitriptyline, nortriptyline and a combination of both (p<0001). The frequencies of CD27 + CD4 + (p<0.05) and CD27 + CD8 + (p<0.01) T-cells were also significantly lower following combination drug treatment. Significantly lower frequencies of IFN-γ-producing CD8 + T-cells were observed with all treatment combinations(p<0.05) and frequencies of IL-17-producing CD4 + and CD8 + T-cells were significantly lower following amitriptyline treatment (p<0.05). Frequencies of Natural Killer T-cells were significantly higher following treatment with nortriptyline (p<0.05). Significantly higher levels of IL-16 (p<0.001) and lower levels of TNF-β (p<0.05) were observed in supernatants. This data indicates that both amitriptyline and nortriptyline modulate the phenotype and function of T-cells and this may have clinical relevance in the pathologies of its off-label applications., (Copyright © 2019 Elsevier B.V. and ECNP. All rights reserved.)

The incidence of oesophageal adenocarcinoma (OAC), arising from reflux-induced Barrett oesophagus (BO), is increasing dramatically. T-cells have recently been implicated in the initiation of oesophagitis; however, their role in the progression from oesophagitis to BO and OAC has not been fully elucidated. Previous studies have examined the secreted cytokines from oesophageal tissue during disease progression but this study is the first to examine the activation phenotype and the inflammatory profile of CD4(+) and CD8(+) T-cells in human oesophagitis, BO and OAC tissue. Results demonstrated significantly higher levels of IL-4 producing CD4(+) T-cells and secreted levels of IL-6, confirming a Th2 phenotype in BO. In OAC tissue, both pro- and anti-inflammatory cytokines were secreted, with significantly higher levels of IL-6, IL-1β, TNF-α, IFN-γ, IL-2 and IL-10 compared with normal oesophageal tissue. In addition, CD4(+) T-cells infiltrating OAC tissue displayed a decreased activation profile, with significantly lower CD45RO and CD69 expression compared with normal tissue. Data from this study suggest that factors in the tissue microenvironment may alter T-cell phenotype and function early during oesophageal disease progression and may represent targets for immune intervention., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)