Multicentre harmonisation of a six-colour flow cytometry panel for naïve/memory T cell immunomonitoring

Background. Personalised medicine in oncology needs standardised immunological assays. Flow cytometry (FCM) methods represent an essential tool for immunomonitoring, and their harmonisation is crucial to obtain comparable data in multicentre clinical trials. The objective of this study was to design a harmonisation workflow able to address the most effective issues contributing to intra- and interoperator variabilities in a multicentre project. Methods. The Italian National Institute of Health (Istituto Superiore di Sanita, ISS) managed a multiparametric flow cytometric panel harmonisation among thirteen operators belonging to five clinical and research centres of Lazio region (Italy). The panel was based on a backbone mixture of dried antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, and anti-CCR7) to detect naive/memory T cells, recognised as potential prognostic/predictive immunological biomarkers in cancer immunotherapies. The coordinating centre distributed frozen peripheral blood mononuclear cells (PBMCs) and fresh whole blood (WB) samples from healthy donors, reagents, and Standard Operating Procedures (SOPs) to participants who performed experiments by their own equipment, in order to mimic a real-life scenario. Operators returned raw and locally analysed data to ISS for central analysis and statistical elaboration. Results. Harmonised and reproducible results were obtained by sharing experimental set-up and procedures along with centralising data analysis, leading to a reduction of cross-centre variability for naive/memory subset frequencies particularly in the whole blood setting. Conclusion. Our experimental and analytical working process proved to be suitable for the harmonisation of FCM assays in a multicentre setting, where high-quality data are required to evaluate potential immunological markers, which may contribute to select better therapeutic options

Identificazione delle isoforme di PrP fisiologica e patologica in arvicole rossastre infettate con diversi ceppi di prioni

The Transmissible Spongiform Encephalopathies (TSE) are a group of rare and lethal neurodegenerative diseases, caused by the conformational conversion of the host’s physiological prion protein (PrPC) in a pathological isoform (PrPSc). TSEs exist as different strains that generate an extraordinary clinical and pathological variety in term of incubation time, involved cells and brain areas. Despite that, they are all characterised by a shared pathogenesis, which invariably lead to PrPScaccumulation and to the development of the neuropathologic “triad”. The neurotoxicity mechanisms of prion diseases are not yet known, although all the evidences so far point to the quantitative and conformational variations of PrP. The main hypotheses suggest that a loss of function, that is the loss of the of physiological PrP functions (PrPC), an endogen proteolytic alteration of the PrPC or the acquisition of new functions from the pathological PrP (gain of function) can be the cause of the neurotoxicity encountered in the EST. To properly tackle these hypotheses, it is necessary a better understanding of the phenomena that involves the PrP during the prion disease, following both, the modifications of the PrPC isoforms and their trans-conformation in PrPSc. Previous studies focusing on these issues used few strains or one only monoclonal antibody, obtaining limited and partially contradictory results either for the use of low-resolution methods and for the low biological and biochemical variability of the strains studied. With the aim to deepen our knowledge on these issues, we selected seven TSE strains extremely variable for source (human and animal), incubation period, biochemical and pathological characteristics. In addition, a new separation assay has been developed based on the differential solubility between PrPC and PrPSc, following the example of the one introduced by this lab (Pirisinu et al., 2013), but optimised in order to get the best experimental conditions for the PrPC and PrPSc separation. Finally, by combining the solubility separation principle with the deglycosylation enzymatic assay and by using different mAbs that identify specific epitopes along the PrP, this project has conceived epitope-mapping techniques both in solution, through western blot, and in situ, through discriminative IHC. These techniques allowed to identify the PrPC and PrPSc isoforms in solution and localize the main PrPSc isoforms in the pathological tissue, including the specific identification of the shedded isoforms. This methodology, even being relatively rapid, fully exploits the different solubility characteristics of the two isoforms and allowed parallel analyses of PrPC and PrPSc in many samples, where the PrPC, being in monomeric form, is in the soluble fraction (supernatant) while the PrPSc, in aggregated form, is in the insoluble fraction (pellet). Concerning PrPC, on the basis of previous evidences of its important downregulation during prion diseases, we studied its overall expression level as well as the relative variation of its isoforms. Despite it has been observed in some strains a drastic statistical reduction of the PrPC in the CNS of the infected animals, we did not identify downregulation of PrPC as shared phenomenon for all the strains investigated, being less evident or even absent in the half of them. The results obtained on the single isoforms suggest that the PrPC decrease, when present, is mostly dependent on the full-length PrPC. At variance, for the strains in which PrPC downregulation was not identified, we found that the physiological regulation of the different PrPC isoform is unchanged with respect to the physiological conditions. These results do not support the hypotheses for which a loss of function or an alteration of the proteolytic events impacting the PrPC can represent a determinant role in the neurotoxicity induction from prions, although we can not exclude a role in the pathological context of some specific strains. Concerning PrPSc, at first, we identified the strain-specific isoforms, which showed an extremely variable scenario among the strains, corroborating the idea that PrPSc conformational differences are at the base of their variability. This not only allowed to study the known isoforms of native PrPSc, but also to identify new ones and analyse, for the first time in quantitative terms, also the shedded isoforms of PrPSc. We then investigated the amount of total PrPSc and of its different isoforms that accumulate in the brain of the sick animals, and how these correlate with the disease incubation period and the type of neurodegenerative pathology observed. By analysing the amount of total PrPSc we observed important differences among strains, whit strains characterized by short incubation period (used in this study as proxy of neurotoxicity) showing lower level of PrPSc accumulation. In this sense, our results do not support the hypothesis for which PrPSc can perform a toxic action by itself. In the contrary, the study observed interesting correlations between the incubation time of the different strains and the amount of shedded PrPSc and of fragments truncated at the N-terminus (overall defined as CTF) relative to the full-length isoform anchored on the cell surface. The strains in which these phenomena were more pronounced show a minor neurotoxicity (longer survival period) with respect to those in which these pathological events were less evident. This suggests a possible neuroprotective role of these isoforms or of the cells’ reactions phenomena involved in their production. Then we investigated the in situ localization of these PrPSc isoforms, determining that the shedded isoforms of PrPSc accumulate in the extra-cellular space while the CTF were detected at intracellular level, probably in lysosomes where the N-terminus of PrPSc, protease sensitive, is trimmed by lysosomal enzymes. The full-length isoform of PrPSc is instead anchored at the plasma membrane. Overall, our results lead to hypothesize that the main neurotoxic trigger can be represented by the accumulation of PrPSc on the cell membrane, also involving a decrease of PrPC in some strains. In this context, the shedding and the endocytosis of PrPSc may represent cellular reactions able to play a neuroprotective role, since they cause PrPSc dismissal from the neuron cell surface and, in the case of endocytosis, a partial PrPSc degradation. In conclusion, this project contributes to highlight two possible therapeutics targets for prion diseases, represented by strategies aiming at increasing shedding and/or autophagy of the PrPSc in the affected cellular populations

An optimized Western blot assay provides a comprehensive assessment of the physiological endoproteolytic processing of the prion protein

15 Pág.The prion protein (PrPC) is subjected to several conserved endoproteolytic events producing bioactive fragments that are of increasing interest for their physiological functions and their implication in the pathogenesis of prion diseases and other neurodegenerative diseases. However, systematic and comprehensive investigations on the full spectrum of PrPC proteoforms have been hampered by the lack of methods able to identify all PrPC-derived proteoforms. Building on previous knowledge of PrPC endoproteolytic processing, we thus developed an optimized Western blot assay able to obtain the maximum information about PrPC constitutive processing and the relative abundance of PrPC proteoforms in a complex biological sample. This approach led to the concurrent identification of the whole spectrum of known endoproteolytic-derived PrPC proteoforms in brain homogenates, including C-terminal, N-terminal and, most importantly, shed PrPC-derived fragments. Endoproteolytic processing of PrPC was remarkably similar in the brain of widely used wild type and transgenic rodent models, with α-cleavage-derived C1 representing the most abundant proteoform and ADAM10-mediated shedding being an unexpectedly prominent proteolytic event. Interestingly, the relative amount of shed PrPC was higher in WT mice than in most other models. Our results indicate that constitutive endoproteolytic processing of PrPC is not affected by PrPC overexpression or host factors other than PrPC but can be impacted by PrPC primary structure. Finally, this method represents a crucial step in gaining insight into pathophysiological roles, biomarker suitability, and therapeutic potential of shed PrPC and for a comprehensive appraisal of PrPC proteoforms in therapies, drug screening, or in the progression of neurodegenerative diseases.H. C. A. was supported by the CJD Foundation, Inc and Alzheimer Forschung Initiative e.V. (grant no.: 19050p); J. C. was supported by Spanish Ministry of Science award (grant no.: PID2021-122201OB-C21) cofunded by European Regional Development Fund. R. N. was supported by the Ministero della Salute (grant no.: RF-2016-02364498).Peer reviewe

Blood immune cells as potential biomarkers predicting relapse-free survival of stage III/IV resected melanoma patients treated with peptide-based vaccination and interferon-alpha

IntroductionDespite the recent approval of several therapies in the adjuvant setting of melanoma, tumor relapse still occurs in a significant number of completely resected stage III-IV patients. In this context, the use of cancer vaccines is still relevant and may increase the response to immune checkpoint inhibitors. We previously demonstrated safety, immunogenicity and preliminary evidence of clinical efficacy in stage III/IV resected melanoma patients subjected to a combination therapy based on peptide vaccination together with intermittent low-dose interferon-α2b, with or without dacarbazine preconditioning (https://www.clinicaltrialsregister.eu/ctr-search/search, identifier: 2008-008211-26). In this setting, we then focused on pre-treatment patient immune status to highlight possible factors associated with clinical outcome.MethodsMultiparametric flow cytometry was used to identify baseline immune profiles in patients’ peripheral blood mononuclear cells and correlation with the patient clinical outcome. Receiver operating characteristic curve, Kaplan-Meier survival and principal component analyses were used to evaluate the predictive power of the identified markers.ResultsWe identified 12 different circulating T and NK cell subsets with significant (p ≤ 0.05) differential baseline levels in patients who later relapsed with respect to patients who remained free of disease. All 12 parameters showed a good prognostic accuracy (AUC>0.7, p ≤ 0.05) and 11 of them significantly predicted the relapse-free survival. Remarkably, 3 classifiers also predicted the overall survival. Focusing on immune cell subsets that can be analyzed through simple surface staining, three subsets were identified, namely regulatory T cells, CD56dimCD16- NK cells and central memory γδ T cells. Each subset showed an AUC>0.8 and principal component analysis significantly grouped relapsing and non-relapsing patients (p=0.034). These three subsets were used to calculate a combination score that was able to perfectly distinguish relapsing and non-relapsing patients (AUC=1; p=0). Noticeably, patients with a combined score ≥2 demonstrated a strong advantage in both relapse-free (p=0.002) and overall (p=0.011) survival as compared to patients with a score <2.DiscussionPredictive markers may be used to guide patient selection for personalized therapies and/or improve follow-up strategies. This study provides preliminary evidence on the identification of peripheral blood immune biomarkers potentially capable of predicting the clinical response to combined vaccine-based adjuvant therapies in melanoma