Probes for INS

Abstract

BACKGROUND & AIM:

γδ T-cells comprise a substantial proportion of tissue-associated lymphocytes. However, our current understanding of human γδ T-cells is primarily based on peripheral blood subsets, while the immunobiology of tissue-associated subsets remains largely unclear.

METHODS:

To address this, we characterised the TCR diversity, immunophenotype and function of human liver infiltrating γδ T-cells, focussing on the predominant tissue-associated Vδ2neg γδ subset, which is implicated in liver immunopathology.

RESULTS:

Intrahepatic Vδ2neg γδ T-cells were highly clonally focussed, with single expanded clonotypes featuring complex, private TCR rearrangements frequently dominating the compartment. Such T-cells were predominantly CD27lo/neg effector lymphocytes, whereas naïve CD27hi, TCR diverse populations present in matched blood were generally absent in the liver. Furthermore, while a CD45RAhi Vδ2neg γδ effector subset present in both liver and peripheral blood contained overlapping TCR clonotypes, the liver Vδ2neg γδ T-cell pool also included a phenotypically distinct CD45RAlo effector compartment that was enriched for expression of the tissue tropism marker CD69, the hepatic homing chemokine receptors CXCR3 and CXCR6, and liver-restricted TCR clonotypes, suggestive of intrahepatic tissue residency. Liver infiltrating Vδ2neg γδ cells were capable of polyfunctional cytokine secretion, and unlike peripheral blood subsets, were responsive to both TCR and innate stimuli.

CONCLUSION:

These findings suggest the ability of Vδ2neg γδ T-cells to undergo clonotypic expansion and differentiation is crucial in permitting access to solid tissues such as the liver, and can result in functionally distinct peripheral and liver-resident memory γδ T-cell subsets. They highlight the inherent functional plasticity within the Vδ2neg γδ T-cell compartment, and may inform design of cellular therapies involving intrahepatic trafficking of γδ T-cells to suppress liver inflammation or combat liver cancer.

LAY SUMMARY:

γδ T cells are frequently enriched in many solid tissues, however the immunobiology of such tissue-associated subsets in humans has remained unclear. We show that intrahepatic γδ T cells are enriched for clonally expanded effector T cells, whereas naïve γδ T cells are largely excluded; moreover, whereas a distinct proportion of circulating T cell clonotypes was present in both the liver tissue and peripheral blood, a functionally and clonotypically distinct population of liver-resident γδ T cells was also evident. Our findings suggest that factors triggering γδ T cell clonal selection and differentiation, such as infection, can drive enrichment of γδ T cells into liver tissue, allowing the development of functionally distinct tissue-restricted memory populations specialised in local hepatic immunosurveillance.

Routine testing for p16 immunohistochemistry (with selective HPV-specific test use) has been recommended for clinical practice in oropharyngeal squamous cell carcinoma (OPSCC). Data suggests that the E6H4 clone performs best for this purpose, yet no studies have evaluated the optimal antibody concentration for OPSCC testing. We evaluated three concentrations (undiluted, 1:5, and 1:10) of the primary antibody solution for E6H4 using tissue microarrays from a cohort of 199 OPSCC patients with a > 70% staining cutoff for positivity. Concordance was evaluated using percent agreement and Cohen's kappa. The concentrations were evaluated for sensitivity and specificity using high risk HPV RNA in situ hybridization (RNA-ISH) and also correlated with Kaplan-Meier overall survival analysis. Inter-rater agreement was very high between p16 results at each concentration and also with RNA in situ hybridization (p < 0.0001 for all). Agreement between p16 undiluted and 1:5 dilution (agreement 98.2%; Kappa 0.943; p < 0.0001) was very high and between p16 undiluted and 1:10 dilution (agreement 79.2%; Kappa 0.512; p < 0.0001) much lower. Intensity of the staining did decrease with the 1:5 and 1:10 dilutions compared to undiluted, but not in a manner that obviously would change test interpretation or performance. Results suggest that the E6H4 antibody performs well at dilutions of up to 1:5 fold with a minor decrease in staining intensity, minimum loss of sensitivity, and no loss of specificity in OPSCC patients. This could result in reagent and cost savings.

Recently three different cyclin-dependent kinase 4 and 6 (CDK4/6) dual inhibitors were approved for the treatment of breast cancer (palbociclib, ribociclib and abemaciclib), all of which offer comparable therapeutic benefits. Their safety profiles however are different. For example, neutropenia is observed at varying incidences in patients treated with these drugs; however it is the most common adverse event for palbociclib and ribociclib, whereas diarrhea is the most common adverse event observed in patients treated with abemaciclib. In order to understand the mechanism of diarrhea observed with these drugs and in an effort to guide the development of safer drugs, we compared the effects of oral administration of palbociclib, ribociclib and abemaciclib on the gastrointestinal tract of rats using doses intended to produce comparable CDK4/6 inhibition. Rats administered abemaciclib, but not palbociclib or ribociclib, had fecal alterations, unique histopathological findings and distinctive changes in intestinal gene expression. Morphologic changes in the intestine were characterized by proliferation of crypt cells, loss of goblet cells, poorly differentiated and degenerating enterocytes with loss of microvilli and mucosal inflammation. In the jejunum of abemaciclib-treated rats, down-regulation of enterocyte membrane transporters and up-regulation of genes associated with cell proliferation were observed, consistent with activation of the Wnt pathway and downstream transcriptional regulation. Among these CDK4/6 inhibitors, intestinal toxicity was unique to rats treated with abemaciclib, suggesting a mechanism of toxicity not due to primary pharmacology (CDK4/6 inhibition), but to activity at secondary pharmacological targets.

The extent of surgical resection is significantly correlated with outcome in glioma; however, current intraoperative navigational tools are useful only in a subset of patients. We show here that a new optical intraoperative technique, Cerenkov luminescence imaging (CLI) following intravenous injection of O‑(2-[18F]fluoroethyl)-L-tyrosine (FET), can be used to accurately delineate glioma margins, performing better than the current standard of fluorescence imaging with 5-aminolevulinic acid (5-ALA).

Methods: Rats implanted orthotopically with U87, F98 and C6 glioblastoma cells were injected with FET and 5-aminolevulinic acid (5-ALA). Positive and negative tumor regions on histopathology were compared with CL and fluorescence images. The capability of FET CLI and 5-ALA fluorescence imaging to detect tumor was assessed using receptor operator characteristic curves and optimal thresholds (CLIOptROC and 5-ALAOptROC) separating tumor from healthy brain tissue were determined. These thresholds were used to guide prospective tumor resections, where the presence of tumor cells in the resected material and in the remaining brain were assessed by Ki-67 staining.

Results: FET CLI signal was correlated with signal in preoperative PET images (y = 1.06x - 0.01; p < 0.0001) and with expression of the amino acid transporter SLC7A5 (LAT1). FET CLI (AUC = 97%) discriminated between glioblastoma and normal brain in human and rat orthografts more accurately than 5-ALA fluorescence (AUC = 91%), with a sensitivity >92% and specificity >91%, and resulted in a more complete tumor resection.

Conclusion: FET CLI can be used to accurately delineate glioblastoma tumor margins, performing better than the current standard of fluorescence imaging following 5-ALA administration, and is therefore a promising technique for clinical translation.

The Caribbean spiny lobster Panulirus argus is susceptible to infection by Panulirus argus Virus 1 (PaV1), the only virus known to naturally infect any lobster species. However, P. argus is able to mitigate PaV1 transmission risk by avoiding infected individuals. P. argus may also be susceptible to another lethal virus, White Spot Syndrome Virus (WSSV). WSSV has not been documented in wild populations of spiny lobsters, but has been experimentally transmitted to six other lobster species from the genus Panulirus. Although WSSV has been detected intermittently in wild populations of shrimp in the Caribbean region, the risk to P. argus has not been evaluated. Potential emergence of the disease could result in fisheries losses and ecological disruption. To assess the risk to P. argus, we tested its susceptibility to WSSV via injection and waterborne transmission. We also tested whether healthy lobsters can detect and avoid conspecifics with qPCR-quantifiable WSSV infections. We found P. argus to be highly susceptible to WSSV via intramuscular injection, with mortality reaching 88% four weeks post inoculation. Panulirus argus was also susceptible to WSSV via waterborne transmission, but WSSV burden was low after four weeks via qPCR. Behavioral assays indicated that P. argus can detect and avoid conspecifics infected with WSSV and the avoidance response was strongest for the most heavily infected individuals - a response comparable to PaV1-infected conspecifics. Panulirus argus is the first spiny lobster found to be susceptible to WSSV in the Americas, but it is possible that a generalized avoidance response by healthy lobsters against infected conspecifics provides a behavioral defense and may reduce WSSV infection potential and prevalence. Preliminary evidence suggests that the infiltration of hemolymph constituents into the urine may be the source of the avoidance behavior and could therefore extend to other directly transmitted pathogens in spiny lobster populations preventing them from becoming common in their population.

Four immunohistochemistry (IHC) diagnostic assays have been approved for tumour PD-L1 protein assessment in the clinic. However, mRNA detection by in situ hybridisation (ISH) could be utilised as an alternative to protein detection. Detecting spatial changes in gene expression provides vital prognostic and diagnostic information, particularly in immune oncology where the phenotype, cellular infiltration and immune activity status may be associated with patient survival. Translation of mRNA expression to a clinically relevant cut off or threshold is challenging due to variability between assays and the detection of different analytes. These studies aim to confirm the suitability of formalin fixed paraffin embedded (FFPE) tissue sections for use with RNA ISH. A comparison of mRNA expression and protein expression may inform the suitability of mRNA as a patient selection biomarker in a similar manner to IHC and provide evidence of a suitable scoring algorithm. Ninety patient samples, thirty for each indication of non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC) and urothelial carcinoma (UC), previously assessed using the VENTANA PD-L1 (SP263) Assay were chosen to represent a wide dynamic range of percentage tumour cell staining (TCIHC). Expression of mRNA was assessed by ISH using the RNAScope 2.5 assay and probe CD274/PD-L1 (Advanced Cell Diagnostics) including kit provided positive and negative control probes. Brightfield whole slide images of tissues were captured. The percentage of tumour cells with PD-L1 mRNA expression (%TCmRNA) and mean punctate dots/tumour cell were determined using image analysis. Differences in RNA expression between the IHC derived TCIHC≥25% and <25% groups were assessed using t-tests. For each indication, a receiver-operating characteristic (ROC) analysis identified thresholds for patient classification using %TCmRNA and dots/tumour cell, with reference to TCIHC≥25%. Eighty-six samples were successfully tested; 3 failed due to insufficient control probe staining, 1 due to lack of tumour. Percent TCmRNA staining using RNAScope demonstrated statistical significance (at α = 0.05) in the PD-L1 high (TCIHC ≥25%) vs the PD-L1 low (TCIHC <25%) groups for NSCLC, HNSCC, and UC. The number of punctate dots/tumour cell was significantly higher in the PD-L1 high vs the PD-L1 low groups for NSCLC and HNSCC but not UC. For %TCmRNA; ROC analysis identified thresholds of: NSCLC 18.0%, HNSCC 31.8%, UC 25.8%. For dots/tumour cell, thresholds were: NSCLC 0.26, HNSCC 0.53, UC 0.45. Routine tissue fixation and processing is suitable for RNA detection using RNAScope. PD-L1 mRNA extent and level is associated with PD-L1 status determined by IHC. Threshold optimisation for %TCmRNA and mean dots/tumour cell results in high specificity to IHC PD-L1 classification, but only moderate sensitivity.