Non-terminally exhausted tumor-resident memory HBV-specific T cell responses correlate with relapse-free survival in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) often develops following chronic hepatitis B virus (HBV) infection and responds poorly to immune checkpoint blockade. Here, we examined the antigen specificities of HCC-infiltrating T cells and their relevance to tumor control. Using highly multiplexed peptide-MHC tetramer staining of unexpanded cells from blood, liver, and tumor tissues from 46 HCC patients, we detected 91 different antigen-specific CD8+ T cell populations targeting HBV, neoantigen, tumor-associated, and disease-unrelated antigens. Parallel high-dimensional analysis delineated five distinct antigen-specific tissue-resident memory T (Trm) cell populations. Intratumoral and intrahepatic HBV-specific T cells were enriched for two Trm cell subsets that were PD-1loTOXlo, despite being clonally expanded. High frequencies of intratumoral terminally exhausted T cells were uncommon. Patients with tumor-infiltrating HBV-specific CD8+ Trm cells exhibited longer-term relapse-free survival. Thus, non-terminally exhausted HBV-specific CD8+ Trm cells show hallmarks of active involvement and effective antitumor response, implying that these cells could be harnessed for therapeutic purposes.

A High-Throughput Colorimetric Microplate Assay for Determination of Plasma Arginase Activity.

Arginase, an enzyme involved in the urea cycle, is gaining attention as a critical player in numerous chronic pathologies. Additionally, increased activity of this enzyme has been shown to correlate with poor prognosis in a range of cancers. Colorimetric assays that measure the conversion of arginine to ornithine have long been used to determine the activity of arginase. However, this analysis is hindered by a lack of standardization across protocols. Here, we describe in detail a novel revision of the Chinard's colorimetric assay used to determine arginase activity. Dilution series of patient plasma are plotted to form a logistic function, from which activity can be interpolated by comparison to an ornithine standard curve. Inclusion of patient dilution series rather than a single point increases the robustness of the assay. This high-throughput microplate assay analyzes 10 samples per plate to produce highly reproducible results.

Mass cytometry analysis reveals altered immune profiles in patients with coronary artery disease.

Objective: The importance of inflammation in atherosclerosis is well accepted, but the role of the adaptive immune system is not yet fully understood. To further explore this, we assessed the circulating immune cell profile of patients with coronary artery disease (CAD) to identify discriminatory features by mass cytometry. Methods: Mass cytometry was performed on patient samples from the BioHEART-CT study, gated to detect 82 distinct cell subsets. CT coronary angiograms were analysed to categorise patients as having CAD (CAD+) or having normal coronary arteries (CAD-). Results: The discovery cohort included 117 patients (mean age 61 ± 12 years, 49% female); 79 patients (68%) were CAD+. Mass cytometry identified changes in 15 T-cell subsets, with higher numbers of proliferating, highly differentiated and cytotoxic cells and decreases in naïve T cells. Five T-regulatory subsets were related to an age and gender-independent increase in the odds of CAD incidence when expressing CCR2 (OR 1.12), CCR4 (OR 1.08), CD38 and CD45RO (OR 1.13), HLA-DR (OR 1.06) and Ki67 (OR 1.22). Markers of proliferation and differentiation were also increased within B cells, while plasmacytoid dendritic cells were decreased. This combination of changes was assessed using SVM models in discovery and validation cohorts (area under the curve = 0.74 for both), confirming the robust nature of the immune signature detected. Conclusion: We identified differences within immune subpopulations of CAD+ patients which are indicative of a systemic immune response to coronary atherosclerosis. This immune signature needs further study via incorporation into risk scoring tools for the precision diagnosis of CAD.

Effects of storage time and temperature on highly multiparametric flow analysis of peripheral blood samples; implications for clinical trial samples.

We sought to determine the effect of time and temperature of blood sample storage before preparation of human peripheral blood mononuclear cells (PBMCs) by Ficoll-hypaque density gradient centrifugation. Blood samples from healthy donors were stored at room temperature (RT) or refrigerated at 4°C before preparation of PBMCs. Cell yield and viability, and proportions of major cell populations within PBMCs, as determined by fluorescence flow cytometry, were assessed for both fresh and cryopreserved samples. Highly multiparametric mass cytometry was performed on cryopreserved PBMCs. We found that refrigeration had marked negative effects on subsequent PBMC yield. Storage at RT led to co-purification of low density neutrophils with PBMCs, but had no detectable effects on the proportions of multiple cell subsets including, but not limited to, monocytes, NK cells, B cells, Treg cells, and naïve, central memory and effector memory CD4+ and CD8+ T cells and CD45RA-positive terminal effector CD8+ T cells. Expression of a number of cell surface receptors, including CXCR5, CCR6, CXCR3 and TIGIT, but not CD247 was reduced after RT storage before PBMC preparation, and this effect correlated with the degree of low density neutrophil contamination. As such, when PBMC preparation cannot be undertaken immediately after blood draw, storage at RT is far superior to refrigeration. RT storage leads to neutrophil activation, but does not compromise measurement of PBMC subset distribution. However caution must be applied to interpretation of cytometric measurements of surface molecules such as chemokine receptors.

P53 activation suppresses irinotecan metabolite SN-38-induced cell damage in non-malignant but not malignant epithelial colonic cells.

Nutlin-3a is a p53 activator and potential cyclotherapy approach that may also mitigate side effects of chemotherapeutic drugs in the treatment of colorectal cancer. We investigated cell proliferation in a panel of colorectal cancer (CRC) cell lines with wild-type or mutant p53, as well as a non-tumorigenic fetal intestinal cell line following Nutlin-3a treatment (10 µM). We then assessed apoptosis at 24 and 48 h following administration of the active irinotecan metabolite, SN-38 (0.001 µM - 1 µM), alone or following pre-treatment with Nutlin-3a (10 µM). Nutlin-3a treatment (10 µM) significantly reduced proliferation in wild-type p53 expressing cell lines (FHS 74 and HCT116+/+) at 72 and 96 h, but was without effect in cell lines with mutated or deleted p53 (Caco-2, SW480, and HCT 116-/-). SN-38 treatment induced significant apoptosis in all cell lines after 48 h. Nutlin-3a unexpectedly increased cell death in the p53 wild-type CRC cell line, HCT116+/+, while Nutlin-3a pre-treatment provided protection from SN-38 in the p53 wild-type normal cell line, FHs 74. These results demonstrate Nutlin-3a's selective growth-arresting efficacy in p53 wild-type non-malignant intestinal cell lines, enabling the selective targeting of malignant cells with chemotherapy drugs. These studies highlight the potential of Nutlin-3a to minimise intestinal mucosal damage following chemotherapy.

Author Correction: Lung endothelial cell antigen cross-presentation to CD8+T cells drives malaria-associated lung injury.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Lung endothelial cell antigen cross-presentation to CD8+T cells drives malaria-associated lung injury.

Malaria-associated acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are life-threatening manifestations of severe malaria infections. The pathogenic mechanisms that lead to respiratory complications, such as vascular leakage, remain unclear. Here, we confirm that depleting CD8+T cells with anti-CD8ß antibodies in C57BL/6 mice infected with P. berghei ANKA (PbA) prevent pulmonary vascular leakage. When we transfer activated parasite-specific CD8+T cells into PbA-infected TCRß-/- mice (devoid of all T-cell populations), pulmonary vascular leakage recapitulates. Additionally, we demonstrate that PbA-infected erythrocyte accumulation leads to lung endothelial cell cross-presentation of parasite antigen to CD8+T cells in an IFNγ-dependent manner. In conclusion, pulmonary vascular damage in ALI is a consequence of IFNγ-activated lung endothelial cells capturing, processing, and cross-presenting malaria parasite antigen to specific CD8+T cells induced during infection. The mechanistic understanding of the immunopathogenesis in malaria-associated ARDS and ALI provide the basis for development of adjunct treatments.

Mutually exclusive FGFR2, HER2, and KRAS gene amplifications in gastric cancer revealed by multicolour FISH.

Gastric cancer (GC) is a major cause of global cancer mortality. Previous genomic studies have reported that several RTK-RAS pathway components are amplified in GC, with individual tumours often amplifying one component and not others ("mutual exclusivity"). Here, we sought to validate these findings for three RTK/RAS components (FGFR2, HER2, KRAS) using fluorescence in situ hybridisation (FISH) on a series of gastric tumours, cell lines and patient-derived xenografts. Applying dual-colour FISH on 137 gastric tumours (89 FFPE surgical resections and 48 diagnostic biopsies), we observed FGFR2 amplification in 7.3% and HER2 amplification in 2.2% of GCs. GCs exhibiting FGFR2 amplification were associated with high tumour grade (p = 0.034). In FISH positive tumours, striking differences in copy number levels between cancer cells in the same tumour were observed, suggesting intra-tumour heterogeneity. Using a multicolour FISH assay allowing simultaneous detection of FGFR2, HER2, and KRAS amplifications, we confirmed that these components exhibited a mutually exclusive pattern of gene amplification across patients. The FISH data were also strongly correlated with Q-PCR levels and at the protein level by immunohistochemistry. Our data confirm that RTK/RAS components are mutually exclusively amplified in GC, and demonstrate the feasibility of identifying multiple aneuploidies using a single FISH assay. Application of this assay to GC samples, particularly diagnostic biopsies, may facilitate enrollment of GC patients into clinical trials evaluating RTK/RAS directed therapies. However, the presence of intra-tumour heterogeneity may require multiple biopsy samples to be obtained per patient before a definitive diagnosis can be attained.