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Background We evaluated the patterns of peripheral Toll-like receptor (TLR) signaling activity and the expression of TLRs and natural killer (NK) cell activation in a cohort of patients experiencing severe hepatitis flares after stopping nucleot(s)ide analogues (NAs) therapy. Methods Samples were collected longitudinally from patients with chronic hepatitis B who were enrolled in a prospective study of NA discontinuation. Patients experiencing hepatitis flares were compared with patients with normal alanine aminotransferase. Peripheral blood mononuclear cells (PBMCs) were stimulated with TLR ligands and cytokine secretion in the cell culture supernatant measured. Expression of TLR2/4, NKG2D, NKp46, and triggering receptor expressed on myeloid cells 1 (TREM-1) on monocytes, NK, and NK-T cells was measured. Results Seventeen patients with severe reactivation hepatitis flares were compared to 12 nonflare patients. Hepatitis flares were associated with increased activity of TLR2–8 and TLR9 signaling in PBMCs at the time of peak flare compared to baseline. Hepatitis flares were also associated with (1) upregulation of TLR2 and (2) TREM-1 receptor expression on NK. There were no differences at baseline between flare patients and nonflare patients. Conclusions Hepatitis flares off NA therapy have a significant innate inflammatory response with upregulation of TLR signaling on peripheral monocytes and TLR2 and TREM-1 expression on NK cells. This implicates the innate immune system in the immunopathogenesis of hepatitis B flares.

Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a ‘super engager’ behavioral cluster comprising T cells with potent serial killing capacity. Among other T cell concepts we also study cancer metabolome-sensing engineered T cells (TEGs) and detect behavior-specific gene signatures that include a group of 27 genes with no previously described T cell function that are expressed by super engager killer TEGs. We further show that type I interferon can prime resistant organoids for TEG-mediated killing. BEHAV3D is a promising tool for the characterization of behavioral-phenotypic heterogeneity of cellular immunotherapies and may support the optimization of personalized solid-tumor-targeting cell therapies.

Background: Organoids are in vitro three-dimensional structures that can be grown from patient tissue. Head and neck cancer (HNC) is a collective term used for multiple tumor types including squamous cell carcinomas and salivary gland adenocarcinomas.Methods: Organoids were established from HNC patient tumor tissue and characterized using immunohistochemistry and DNA sequencing. Organoids were exposed to chemo- and radiotherapy and a panel of targeted agents. Organoid response was correlated with patient clinical response. CRISPR-Cas9-based gene editing of organoids was applied for biomarker validation.Findings: A HNC biobank consisting of 110 models, including 65 tumor models, was generated. Organoids retained DNA alterations found in HNC. Comparison of organoid and patient response to radiotherapy (primary [n = 6] and adjuvant [n = 15]) indicated potential for guiding treatment options in the adjuvant setting. In organoids, the radio-sensitizing potential of cisplatin and carboplatin could be validated. However, cetuximab conveyed radioprotection in most models. HNC-targeted treatments were tested on 31 models, indicating possible novel treatment options with the potential for treatment stratification in the future. Activating PIK3CA mutations did not predict alpelisib response in organoids. Protein arginine methyltransferase 5 (PRMT5) inhibitors were identified as a potential treatment option for cyclin-dependent kinase inhibitor 2A (CDKN2A) null HNC.Conclusions: Organoids hold potential as a diagnostic tool in personalized medicine for HNC. In vitro organoid response to radiotherapy (RT) showed a trend that mimics clinical response, indicating the predictive potential of patient-derived organoids. Moreover, organoids could be used for biomarker discovery and validation.

We evaluated the patterns of peripheral TLR signalling activity and the expression of TLRs and NK cell activation in a cohort of patients experiencing severe hepatitis flares after stopping NA therapy.Samples were collected longitudinally from CHB patients enrolled in a prospective study of NA discontinuation. Patients experiencing hepatitis flares were compared to patients with normal ALT. PBMC were stimulated with TLR ligands and cytokine secretion in the cell culture supernatant measured. Expression of TLR2/4, NKG2D, NKp46 and TREM-1 on monocytes, NK and NK-T cells was measured.17 patients with severe reactivation hepatitis flares were compared to 12 non-flare patients. Hepatitis flares were associated with increased activity of TLR 2-8 and TLR 9 signalling in PBMC at the time of peak flare compared to baseline. Hepatitis flares were also associated with upregulation of TLR2, and TREM-1 receptor expression on NK. There were no differences at baseline between flare patients and non-flare patients.Hepatitis flares off NA therapy have a significant innate inflammatory response with upregulation of TLR signalling on peripheral monocytes and TLR-2 and TREM-1 expression on NK cells. This implicates the innate immune system in the immunopathogenesis of hepatitis B flares.

Purpose The presence of tumor-infiltrating lymphocytes (TILs) in tumors is superior to conventional pathologic staging in predicting patient outcome. However, their presence does not define TIL functionality. Here we developed an assay that tests TIL cytotoxicity in patients with locally advanced rectal cancer before definitive treatment, identifying those who will obtain a pathologic complete response (pCR). We also used the assay to demonstrate the rescue of TIL function after checkpoint inhibition blockade (CIB). Patients and Methods Thirty-four consecutive patients were identified initially, with successful completion of the assay before surgery in those 17 patients who underwent full treatment. An in vitro cytotoxic assay of rectal cancer tumoroids cocultured with patient-matched TILs was established and validated. Newly diagnosed patients were recruited with pretreatment biopsy specimens processed within 1 month. Evaluation of TIL-mediated tumoroid lysis was performed by measuring the mean fluorescence intensity of cell death marker, propidium iodide. CIB (anti–programmed cell death protein 1 [anti–PD-1] antibody) response was also assessed in a subset of patient specimens. Results Six of the 17 patients achieved an objective pCR on final evaluation of the resected specimen after neoadjuvant chemoradiotherapy. Cytotoxic killing identified the pCR group with a higher mean fluorescence intensity (27,982 [95% CI, 25,340 to 30,625]) compared with the non-pCR cohort (12,428 [95% CI, 9,434 to 15,423]; p < .001). Assessment of the effectiveness of CIB revealed partial restoration of cytotoxicity in TILs with increased PD-1 expression with anti–PD-1 antibody exposure. Conclusion Evaluating TIL function can be undertaken within weeks of the diagnostic biopsy, affording the potential to alter patient management decisions and refine selection for a watch-and-wait protocol. This cytotoxic assay also has the potential to serve as a platform to assist in the additional development of CIB.

The function of MR1-restricted mucosal-associated invariant T (MAIT) cells in tumor immunity is unclear. Here we show that MAIT cell-deficient mice have enhanced NK cell-dependent control of metastatic B16F10 tumor growth relative to control mice. Analyses of this interplay in human tumor samples reveal that high expression of a MAIT cell gene signature negatively impacts the prognostic significance of NK cells. Paradoxically, pre-pulsing tumors with MAIT cell antigens, or activating MAIT cells in vivo, enhances anti-tumor immunity in B16F10 and E0771 mouse tumor models, including in the context of established metastasis. These effects are associated with enhanced NK cell responses and increased expression of both IFN-γ-dependent and inflammatory genes in NK cells. Importantly, activated human MAIT cells also promote the function of NK cells isolated from patient tumor samples. Our results thus describe an activation-dependent, MAIT cell-mediated regulation of NK cells, and suggest a potential therapeutic avenue for cancer treatment., Mucosal-associated invariant T (MAIT) cells facilitate anti-microbial responses, but their functions in cancer protection is unclear. Here the authors show that activated MAIT cells induce an IFN-γ transcriptome in natural killer (NK) cells and enhance NK-dependent anti-cancer immunity in mice, thereby hinting a new avenue for cancer therapy.

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a near-native state. When derived from resected or biopsied tumor tissue, organoids closely recapitulate in vivo tumor morphology and can be used to study therapy response in vitro. Biobanks of tumor organoids reflect the vast variety of clinical tumors and patients and therefore hold great promise for preclinical and clinical applications. This paper presents a method for medium-throughput drug screening using head and neck squamous cell carcinoma and colorectal adenocarcinoma organoids. This approach can easily be adopted for use with any tissue-derived organoid model, both normal and diseased. Methods are described for in vitro exposure of organoids to chemo- and radiotherapy (either as single-treatment modality or in combination). Cell survival after 5 days of drug exposure is assessed by measuring adenosine triphosphate (ATP) levels. Drug sensitivity is measured by the half-maximal inhibitory concentration (IC50), area under the curve (AUC), and growth rate (GR) metrics. These parameters can provide insight into whether an organoid culture is deemed sensitive or resistant to a particular treatment.

Anal cancer is a rare disease that has doubled in incidence over the last four decades. Current treatment and survival of patients with this disease has not changed substantially over this period of time, due, in part, to a paucity of preclinical models to assess new therapeutic options. To address this hiatus, we set-out to establish, validate and characterise a panel of human anal squamous cell carcinoma (ASCC) cell lines by employing an explant technique using fresh human ASCC tumour tissue. The panel of five human ASCC cell lines were validated to confirm their origin, squamous features and tumourigenicity, followed by molecular and genomic (whole-exome sequencing) characterisation. This panel recapitulates the genetic and molecular characteristics previously described in ASCC including phosphoinositide-3-kinase (PI3K) mutations in three of the human papillomavirus (HPV) positive lines and TP53 mutations in the HPV negative line. The cell lines demonstrate the ability to form tumouroids and retain their tumourigenic potential upon xenotransplantation, with varied inducible expression of major histocompatibility complex class I (MHC class I) and Programmed cell death ligand 1 (PD-L1). We observed differential responses to standard chemotherapy, radiotherapy and a PI3K specific molecular targeted agent in vitro, which correlated with the clinical response of the patient tumours from which they were derived. We anticipate this novel panel of human ASCC cell lines will form a valuable resource for future studies into the biology and therapeutics of this rare disease.

MR1-restricted mucosal-associated invariant T (MAIT) cells recognize microbial metabolites and play an important role in immunity to infection, however, the role they play in tumor immunity is unclear. Here we show that MAIT cell-deficient mice are more resistant to subcutaneous and lung metastasis B16F10 tumor growth compared to control mice, an effect that was associated with enhanced NK cell numbers and was NK cell-dependent. Analysis of this interplay in cancer patients also revealed that a high expression of a novel MAIT gene signature negatively impacted the prognostic significance of NK cells. Paradoxically, pre-pulsing tumors with MAIT cell antigens, or antigen-mediated MAIT cell activation in vivo, enhanced immunity against B16F10 and E0771 lung tumor metastasis. Furthermore, MAIT cell activation effectively reduced metastatic burden in a more stringent model of established lung metastases in mice. These effects were associated with enhanced NK cell responses and increased expression of both IFNγ-dependent and inflammatory genes in NK cells, which was neutralized by IFNγ blockade. Importantly, activated human MAIT cells also enhanced the function of NK cells isolated from patient tumor samples. These findings provide insight into the contrasting roles that MAIT cells can play in controlling anti-tumor immune responses depending on their activation status, in both mice and humans, and suggest potential therapeutic avenues for exploiting their potential anti-tumor properties for cancer treatment.

This study investigated the immunomodulatory influence of IL10 producing B regulatory cells, Bregs (CD19+CD24hiCD38hi) to standard Twinrix® vaccination. We also investigated HBsAg specific T-cell mediated IFN-γ responses to Twinrix® which in theory could provide effective immunity despite low anti-HBs titer. A total of 309 hepatitis B negative health care students and workers completed a standard Twinrix® vaccination schedule (0, 1 and 6 months). Depending on the vaccination response the participants were divided in to non-, low- and high responders according to anti-HBs titer (1000 mIU/mL respectively) two months after completed vaccination schedule. Blood samples from baseline and after vaccination from all non- and low-responders (23 participants) and the same number of high-responders were used for flow cytometric analyses of IL10 producing Bregs and T-cell mediated IFN-γ responses. A decrease in levels of IL10 producing Bregs was observed after vaccination in high responders compared to non- and low-responders. Compiling non-and low-responders against high-responders showed a lower T-cell mediated IFN-γ response at baseline in non-and low-responders when stimulated with Engerix® vaccine. In contrary no positive correlation between IL10 producing Bregs or IFN-γ positive T-cells and anti-HBs titer was observed. Hence this study cannot prove that levels of IL10 producing Bregs or IFN-γ positive T cell affect HBV vaccine response.

Background The aim of this study was to monitor the effect of humidified‐warm carbon dioxide (HWCO2) delivered into the open abdomen of mice, simulating laparotomy. Methods Mice were anaesthetized, ventilated and subjected to an abdominal incision followed by wound retraction. In the experimental group, a diffuser device was used to deliver HWCO2; the control group was exposed to passive air flow. In each group of mice, surgical damage was produced on one side of the peritoneal wall. Vital signs and core temperature were monitored throughout the 1‐h procedure. The peritoneum was closed and mice were allowed to recover for 24 h or 10 days. Tumour cells were delivered into half of the mice in each cohort. Tissue was then examined using scanning electron microscopy and immunohistochemistry. Results Passive air flow generated ultrastructural damage including mesothelial cell bulging/retraction and loss of microvilli, as assessed at 24 h. Evidence of surgical damage was still measurable on day 10. HWCO2 maintained normothermia, whereas open surgery alone led to hypothermia. The degree of tissue damage was significantly reduced by HWCO2 compared with that in controls. Peritoneal expression of hypoxia inducible factor 1α and vascular endothelial growth factor A was lowered by HWCO2. These effects were also evident at the surgical damage sites, where protection from tissue trauma extended to 10 days. HWCO2 did not reduce tumorigenesis in surgically damaged sites compared with passive air flow. Conclusion HWCO2 diffusion into the abdomen in the context of open surgery afforded tissue protection and accelerated tissue repair in mice, while preserving normothermia. Surgical relevanceDamage to the peritoneum always occurs during open abdominal surgery, by exposure to desiccating air and by mechanical trauma/damage owing to the surgical intervention. Previous experimental studies showed that humidified‐warm carbon dioxide (HWCO2) reduced peritoneal damage during laparoscopic insufflation. Additionally, this intervention decreased experimental peritoneal carcinomatosis compared with the use of conventional dry‐cold carbon dioxide.In the present experimental study, the simple delivery of HWCO2 into the open abdomen reduced the amount of cellular damage and inflammation, and accelerated tissue repair. Sites of surgical intervention serve as ideal locations for cancer cell adhesion and subsequent tumour formation, but this was not changed measurably by the delivery of HWCO2., Reduced tissue injury

568 Background: Colorectal metastases arise predominantly in the liver, lung and peritoneum. It is becoming increasingly clear that these locations might be distinct in terms of tumor cell tropism and genetics. We explored the immunological landscape in primary colorectal tumors along with hepatic and peritoneal metastases. The ultimate intent is to identify opportunities to implore immunomodulatory strategies to improve patient outcomes. Methods: Cancer biopsies were processed to generate tumor-derived organoids and TILs from patients with primary and/or metastatic tumors. These were co-cultured with patient matched TILs to assess immune competency and opportunities to employ checkpoint blockade using an in vitro platform we recently developed to explore TIL activity in patients with rectal cancer prior to standard of care therapy. Results: We found substantively different levels of TILs, their sub-set composition and most importantly their capacity to kill tumor cells. The nature of the metastatic peritoneal and hepatic tumor TILs were distinct. Where high programmed cell death 1 (PD-1) expression was high on TILs there was an opportunity to test PD-1 receptor function with blocking antibodies whereby some TILs showed enhanced cytotoxicity while others did not. Conclusions: Using our immune function assay we have demonstrated the distinct activities of patient-matched TILs in terms of killing at different organ sites of metastases. We also found differences in response to checkpoint inhibition blockade and de novo immune resistance.

During cancer surgery, prostaglandin-mediated inflammation may promote and activate micrometastatic disease with a consequent increase in long-term cancer recurrence. Cyclooxygenase-2 inhibitors, known to have anti-proliferative properties, may offset such perioperative perturbation. We investigated the effectiveness of these agents to minimize inflammatory changes during cancer surgery.Following ethics approval, 32 patients who were to undergo major intracavity cancer surgery were enrolled in this prospective, randomized, clinical trial. The treatment group received 400 mg celecoxib preoperatively followed by five 200 mg 12-hourly doses. The control group received no anti-inflammatory agents. Inflammatory and immunomodulatory end points were measured serially. The primary end points were the measured plasma and urinary prostaglandin E metabolite (PGENo differences in the 48-hr plasma or urinary PGEStandard dosing of the cyclooxygenase-2 inhibitor celecoxib slightly reduced perioperative cyclooxygenase activity during cancer surgery. Given cyclooxygenase's role in cancer pathways, we recommend dose-finding studies be undertaken before prospective clinical trials are conducted testing the currently unsubstantiated hypothesis that perioperative anti-inflammatory administration improves long-term cancer outcomes. This trial was registered at: Australian New Zealand Clinical Trial Registry: ACTRN12615000041550; www.anzctr.org.au.

The presence of tumor immune infiltrating cells (TILs), particularly CD8(+) T-cells, is a robust predictor of outcome in patients with colorectal cancer (CRC). We revisited TIL abundance specifically in patients with microsatellite stable (MSS) CRC without evidence of lymph node or metastatic spread. Examination of the density of CD8(+) T-cells in primary tumors in the context of other pro-oncogenic markers was performed to investigate potential regulators of TILs. Two independent cohorts of patients with MSS T2-4N0M0 CRC, enriched for cases with atypical relapse, were investigated. We quantified CD8(+) and CD45RO(+) -TILs, inflammatory markers, NFkBp65, pStat3, Cyclo-oxygenase-2 (COX2) and GRP78 as well as transcription factors (TF), β-catenin and MYB. High CD8(+) TILs correlated with a better relapse-free survival in both cohorts (p = 0.002) with MYB and its target gene, GRP78 being higher in the relapse group (p = 0.001); no difference in pSTAT3 and p65 was observed. A mouse CRC (CT26) model was employed to evaluate the effect of MYB on GRP78 expression as well as T-cell infiltration. MYB over-expressing in CT26 cells increased GRP78 expression and the analysis of tumor-draining lymph nodes adjacent to tumors showed reduced T-cell activation. Furthermore, MYB over-expression reduced the efficacy of anti-PD-1 to modulate CT26 tumor growth. This high MYB and GRP78 show a reciprocal relationship with CD8(+) TILs which may be useful refining the prediction of patient outcome. These data reveal a new immunomodulatory function for MYB suggesting a basis for further development of anti-GRP78 and/or anti-MYB therapies.