IMPRINTS.CETSA and IMPRINTS.CETSA.app: an R package and a Shiny application for the analysis and interpretation of IMPRINTS-CETSA data.

IMPRINTS-CETSA (Integrated Modulation of Protein Interaction States-Cellular Thermal Shift Assay) provides a highly resolved means to systematically study the interactions of proteins with other cellular components, including metabolites, nucleic acids and other proteins, at the proteome level, but no freely available and user-friendly data analysis software has been reported. Here, we report IMPRINTS.CETSA, an R package that provides the basic data processing framework for robust analysis of the IMPRINTS-CETSA data format, from preprocessing and normalization to visualization. We also report an accompanying R package, IMPRINTS.CETSA.app, which offers a user-friendly Shiny interface for analysis and interpretation of IMPRINTS-CETSA results, with seamless features such as functional enrichment and mapping to other databases at a single site. For the hit generation part, the diverse behaviors of protein modulations have been typically segregated with a two-measure scoring method, i.e. the abundance and thermal stability changes. We present a new algorithm to classify modulated proteins in IMPRINTS-CETSA experiments by a robust single-measure scoring. In this way, both the numerical changes and the statistical significances of the IMPRINTS information can be visualized on a single plot. The IMPRINTS.CETSA and IMPRINTS.CETSA.app R packages are freely available on GitHub at https://github.com/nkdailingyun/IMPRINTS.CETSA and https://github.com/mgerault/IMPRINTS.CETSA.app, respectively. IMPRINTS.CETSA.app is also available as an executable program at https://zenodo.org/records/10636134.

PDZ scaffolds regulate extracellular vesicle production, composition, and uptake.

Extracellular vesicles (EVs) are membrane-limited organelles mediating cell-to-cell communication in health and disease. EVs are of high medical interest, but their rational use for diagnostics or therapies is restricted by our limited understanding of the molecular mechanisms governing EV biology. Here, we tested whether PDZ proteins, molecular scaffolds that support the formation, transport, and function of signal transduction complexes and that coevolved with multicellularity, may represent important EV regulators. We reveal that the PDZ proteome (ca. 150 proteins in human) establishes a discrete number of direct interactions with the tetraspanins CD9, CD63, and CD81, well-known EV constituents. Strikingly, PDZ proteins interact more extensively with syndecans (SDCs), ubiquitous membrane proteins for which we previously demonstrated an important role in EV biogenesis, loading, and turnover. Nine PDZ proteins were tested in loss-of-function studies. We document that these PDZ proteins regulate both tetraspanins and SDCs, differentially affecting their steady-state levels, subcellular localizations, metabolism, endosomal budding, and accumulations in EVs. Importantly, we also show that PDZ proteins control the levels of heparan sulfate at the cell surface that functions in EV capture. In conclusion, our study establishes that the extensive networking of SDCs, tetraspanins, and PDZ proteins contributes to EV heterogeneity and turnover, highlighting an important piece of the molecular framework governing intracellular trafficking and intercellular communication.

High-dimensional mass cytometry analysis of NK cell alterations in AML identifies a subgroup with adverse clinical outcome.

Natural killer (NK) cells are major antileukemic immune effectors. Leukemic blasts have a negative impact on NK cell function and promote the emergence of phenotypically and functionally impaired NK cells. In the current work, we highlight an accumulation of CD56-CD16+ unconventional NK cells in acute myeloid leukemia (AML), an aberrant subset initially described as being elevated in patients chronically infected with HIV-1. Deep phenotyping of NK cells was performed using peripheral blood from patients with newly diagnosed AML (n = 48, HEMATOBIO cohort, NCT02320656) and healthy subjects (n = 18) by mass cytometry. We showed evidence of a moderate to drastic accumulation of CD56-CD16+ unconventional NK cells in 27% of patients. These NK cells displayed decreased expression of NKG2A as well as the triggering receptors NKp30 and NKp46, in line with previous observations in HIV-infected patients. High-dimensional characterization of these NK cells highlighted a decreased expression of three additional major triggering receptors required for NK cell activation, NKG2D, DNAM-1, and CD96. A high proportion of CD56-CD16+ NK cells at diagnosis was associated with an adverse clinical outcome and decreased overall survival (HR = 0.13; P = 0.0002) and event-free survival (HR = 0.33; P = 0.018) and retained statistical significance in multivariate analysis. Pseudotime analysis of the NK cell compartment highlighted a disruption of the maturation process, with a bifurcation from conventional NK cells toward CD56-CD16+ NK cells. Overall, our data suggest that the accumulation of CD56-CD16+ NK cells may be the consequence of immune escape from innate immunity during AML progression.

Population transcriptogenomics highlights impaired metabolism and small population sizes in tree frogs living in the Chernobyl Exclusion Zone.

BACKGROUND: Individual functional modifications shape the ability of wildlife populations to cope with anthropogenic environmental changes. But instead of adaptive response, human-altered environments can generate a succession of deleterious functional changes leading to the extinction of the population. To study how persistent anthropogenic changes impacted local species' population status, we characterised population structure, genetic diversity and individual response of gene expression in the tree frog Hyla orientalis along a gradient of radioactive contamination around the Chernobyl nuclear power plant. RESULTS: We detected lower effective population size in populations most exposed to ionizing radiation in the Chernobyl Exclusion Zone that is not compensated by migrations from surrounding areas. We also highlighted a decreased body condition of frogs living in the most contaminated area, a distinctive transcriptomics signature and stop-gained mutations in genes involved in energy metabolism. While the association with dose will remain correlational until further experiments, a body of evidence suggests the direct or indirect involvement of radiation exposure in these changes. CONCLUSIONS: Despite ongoing migration and lower total dose rates absorbed than at the time of the accident, our results demonstrate that Hyla orientalis specimens living in the Chernobyl Exclusion Zone are still undergoing deleterious changes, emphasizing the long-term impacts of the nuclear disaster.

Two New Neutrophil Subsets Define a Discriminating Sepsis Signature.

Rationale: Sepsis is the leading cause of death in adult ICUs. At present, sepsis diagnosis relies on nonspecific clinical features. It could transform clinical care to have immune-cell biomarkers that could predict sepsis diagnosis and guide treatment. For decades, neutrophil phenotypes have been studied in sepsis, but a diagnostic cell subset has yet to be identified. Objectives: To identify an early, specific immune signature of sepsis severity that does not overlap with other inflammatory biomarkers and that distinguishes patients with sepsis from those with noninfectious inflammatory syndrome. Methods: Mass cytometry combined with computational high-dimensional data analysis was used to measure 42 markers on whole-blood immune cells from patients with sepsis and control subjects and to automatically and comprehensively characterize circulating immune cells, which enables identification of novel, disease-specific cellular signatures. Measurements and Main Results: Unsupervised analysis of high-dimensional mass cytometry data characterized previously unappreciated heterogeneity within the CD64+ immature neutrophils and revealed two new subsets distinguished by CD123 and PD-L1 (programmed death ligand 1) expression. These immature neutrophils exhibited diminished activation and phagocytosis functions. The proportion of CD123-expressing neutrophils correlated with clinical severity. Conclusions: This study showed that these two new neutrophil subsets were specific to sepsis and detectable through routine flow cytometry by using seven markers. The demonstration here that a simple blood test distinguishes sepsis from other inflammatory conditions represents a key biological milestone that can be immediately translated into improvements in patient care.

Time-limited alterations in cortical activity of a knock-in mouse model of KCNQ2-related developmental and epileptic encephalopathy.

De novo missense variants in the KCNQ2 gene encoding the Kv7.2 subunit of voltage-gated potassium Kv7/M channels are the main cause of developmental and epileptic encephalopathy with neonatal onset. Although seizures usually resolve during development, cognitive/motor deficits persist. To gain a better understanding of the cellular mechanisms underlying network dysfunction and their progression over time, we investigated in vivo, using local field potential recordings of freely moving animals, and ex vivo in layers II/III and V of motor cortical slices, using patch-clamp recordings, the electrophysiological properties of pyramidal cells from a heterozygous knock-in mouse model carrying the Kv7.2 p.T274M pathogenic variant during neonatal, postweaning and juvenile developmental stages. We found that knock-in mice displayed spontaneous seizures preferentially at postweaning rather than at juvenile stages. At the cellular level, the variant led to a reduction in M ​​current density/conductance and to neuronal hyperexcitability. These alterations were observed during the neonatal period in pyramidal cells of layers II/III and during the postweaning stage in pyramidal cells of layer V. Moreover, there was an increase in the frequency of spontaneous network-driven events mediated by GABA receptors, suggesting that the excitability of interneurons was also increased. However, all these alterations were no longer observed in layers II/III and V of juvenile mice. Thus, our data indicate that the action of the variant is regulated developmentally. This raises the possibility that the age-related seizure remission observed in KCNQ2-related developmental and epileptic encephalopathy patients results from a time-limited alteration of Kv7 channel activity and neuronal excitability. KEY POINTS: The electrophysiological impact of the pathogenic c.821C>T mutation of the KCNQ2 gene (p.T274M variant in Kv7.2 subunit) related to developmental and epileptic encephalopathy has been analysed both in vivo and ex vivo in layers II/III and V of motor cortical slices from a knock-in mouse model during development at neonatal, postweaning and juvenile stages. M current density and conductance are decreased and the excitability of layer II/III pyramidal cells is increased in slices from neonatal and postweaning knock-in mice but not from juvenile knock-in mice. M current and excitability of layer V pyramidal cells are impacted in knock-in mice only at the postweaning stage. Spontaneous GABAergic network-driven events can be recorded until the postweaning stage, and their frequency is increased in layers II/III of the knock-in mice. Knock-in mice display spontaneous seizures preferentially at postweaning rather than at juvenile stages.

Reconstitution of natural killer cells in HLA-matched HSCT after reduced-intensity conditioning: impact on clinical outcome.

Recent advances in the development of reduced-intensity conditioning (RIC) have allowed a broader range of patients to access allogeneic hematopoietic stem cell transplantation (HSCT). Reconstitution of an effective immune system post-transplant, including natural killer (NK) cells, is critical for both tumor control and infectious disease control or prevention. The development and functions of NK cells in such settings remain elusive. Here we analyzed NK cell development in HLA-matched HSCT from related or unrelated donors, after RIC that included antithymocyte globulin (N = 45 patients). Our data reveal that NK cells quickly recover after RIC-HSCT, irrespective of donor type. Rapidly re-emerging NK cells, however, remain immature for more than 6 months. Effector functions resemble that of immature NK cells because they poorly produce IFN-γ and TNF-α in response to target cell stimulation, despite a rapid acquisition of degranulation ability and MIP-1ß production. Strikingly, rapid reconstitution of cytokine production correlates with a lower relapse incidence (P = .01) and a better survival rate (P < .0001) at 1 year post-transplant, whereas degranulation capacity was associated with less relapse (P = .05). Our study demonstrates rapid quantitative reconstitution of the NK cell compartment despite administration of potent immune suppressive drugs as part of the conditioning regimen and after transplantation. However, there is a prolonged persistence of functional defects, the correction of which positively correlates with clinical outcome.

DIAgui: a Shiny application to process the output from DIA-NN.

Summary: DIAgui is an R package to simplify the processing of the report file from the DIA-NN software thanks to a Shiny application. It returns the quantification of either the precursors, the peptides, the proteins, or the genes thanks to the MaxLFQ algorithm. In addition, the latest version provides the Top3 and iBAQ quantification and the number of peptides used for the quantification. In the end, DIAgui produces ready-to-interpret files from the results of DIA mass spectrometry analysis and provides visualization and statistical tools that can be used in a user-friendly way. Availability and implementation: Code and documentation are available on GitHub at https://github.com/marseille-proteomique/DIAgui. The package is written in R and also uses C++ code. A vignette shows its use in an R command line workflow.

Low frequency of Vγ9Vδ2 T-cells predicts poor survival in newly diagnosed acute myeloid leukemia.

In several tumor subtypes, increased infiltration of Vγ9Vδ2 T-cells has been shown to have the highest prognostic value compared to other immune subsets. In acute myeloid leukemia (AML), similar findings have been based solely on the inference of transcriptomic data and have not been assessed with respect to confounding factors. This study aimed at determining, by immunophenotypic analysis (flow or mass cytometry) of peripheral blood from AML patients at diagnosis, the prognostic impact of Vγ9Vδ2 T-cell frequency. This was adjusted for potential confounders (age at diagnosis, disease status, European LeukemiaNet classification, leukocytosis, and allogeneic hematopoietic stem cell transplantation as a time-dependent covariate). The cohort was composed of 198 newly diagnosed AML patients. By univariate analysis, patients with lower Vγ9Vδ2 T-cells at diagnosis had significantly lower 5-year overall and relapse-free survivals. These results were confirmed in multivariate analysis (Hazard Ratio [HR]=1.55[1.04-2.30], p=0.030 and HR=1.64[1.06, 2.53], p=0.025). Immunophenotypic alterations observed in patients with lower Vγ9Vδ2 T-cells included a loss of some cytotoxic Vγ9Vδ2 T-cell subsets and a decreased expression of BTN3A on the surface of blasts. Samples expanded regardless of their Vγ9Vδ2 T-cell levels and displayed similar effector functions in vitro. This study confirms the prognostic value of elevated Vγ9Vδ2 T-cells among lymphocytes, in newly diagnosed AML patients. These results provide a strong rationale to consider consolidation protocols aiming at enhancing Vγ9Vδ2 T-cell responses.

Genetic deletion of JAM-C in pre-leukemic cells rewires leukemic stem cell gene expression program in AML.

The leukemic stem cell (LSC) score LSC-17 based on a stemness-related gene expression signature is an indicator of poor disease outcome in acute myeloid leukemia (AML). However, our understanding of the relationships between LSC and pre-leukemic cells is still incomplete. In particular, it is not known whether "niche-anchoring" of pre-leukemic cell affects disease evolution. To address this issue, we conditionally inactivated the adhesion molecule JAM-C expressed by haematopoietic stem cells (HSC) and LSC in an inducible iMLL-AF9-driven AML mouse model. Deletion of Jam3 (encoding JAM-C) before induction of the leukemia-initiating iMLL-AF9 fusion resulted in a shift from long term to short term-HSC expansion, without affecting disease initiation and progression. In vitro experiments showed that JAM-C controlled leukemic cell nesting irrespective of the bone marrow stromal cells used. RNA sequencing performed on leukemic HSC isolated from diseased mice revealed that genes upregulated in Jam3-deficient animals belonged to Activation Protein-1 (AP-1) and TNF-/NFB pathways. Human orthologs of dysregulated genes allowed to identify a score based on AP-1/TNF-a gene expression that was distinct and complementary from LSC-17 score. Sub-stratification of AML patients with LSC-17 and AP-1/TNF-genes signature defined four groups with median survival ranging from below one year to a median not reached after 8 years. Finally, coculture experiments showed that AP-1 activation in leukemic cells was dependent on the nature of stromal cells. Altogether, our results identify the AP-1/TNF- gene signature as a proxy of LSC anchoring in specific bone marrow niches which improves the prognosis value of the LSC-17 score. NCT02320656.

CD25high Effector Regulatory T Cells Hamper Responses to PD-1 Blockade in Triple-Negative Breast Cancer.

Regulatory T cells (Treg) impede effective antitumor immunity. However, the role of Tregs in the clinical outcomes of patients with triple-negative breast cancer (TNBC) remains controversial. Here, we found that an immunosuppressive TNBC microenvironment is marked by an imbalance between effector αßCD8+ T cells and Tregs harboring hallmarks of highly suppressive effector Tregs (eTreg). Intratumoral eTregs strongly expressed PD-1 and persisted in patients with TNBC resistant to PD-1 blockade. Importantly, CD25 was the most selective surface marker of eTregs in primary TNBC and metastases compared with other candidate targets for eTreg depletion currently being evaluated in trials for patients with advanced TNBC. In a syngeneic TNBC model, the use of Fc-optimized, IL2 sparing, anti-CD25 antibodies synergized with PD-1 blockade to promote systemic antitumor immunity and durable tumor growth control by increasing effector αßCD8+ T-cell/Treg ratios in tumors and in the periphery. Together, this study provides the rationale for the clinical translation of anti-CD25 therapy to improve PD-1 blockade responses in patients with TNBC. SIGNIFICANCE: An imbalance between effector CD8+ T cells and CD25high effector Tregs marks immunosuppressive microenvironments in αPD-1-resistant TNBC and can be reversed through effector Treg depletion to increase αPD-1 efficacy.

Anopheles salivary gland proteomes from major malaria vectors.

BACKGROUND: Antibody responses against Anopheles salivary proteins can indicate individual exposure to bites of malaria vectors. The extent to which these salivary proteins are species-specific is not entirely resolved. Thus, a better knowledge of the diversity among salivary protein repertoires from various malaria vector species is necessary to select relevant genus-, subgenus- and/or species-specific salivary antigens. Such antigens could be used for quantitative (mosquito density) and qualitative (mosquito species) immunological evaluation of malaria vectors/host contact. In this study, salivary gland protein repertoires (sialomes) from several Anopheles species were compared using in silico analysis and proteomics. The antigenic diversity of salivary gland proteins among different Anopheles species was also examined. RESULTS: In silico analysis of secreted salivary gland protein sequences retrieved from an NCBInr database of six Anopheles species belonging to the Cellia subgenus (An. gambiae, An. arabiensis, An. stephensi and An. funestus) and Nyssorhynchus subgenus (An. albimanus and An. darlingi) displayed a higher degree of similarity compared to salivary proteins from closely related Anopheles species. Additionally, computational hierarchical clustering allowed identification of genus-, subgenus- and species-specific salivary proteins. Proteomic and immunoblot analyses performed on salivary gland extracts from four Anopheles species (An. gambiae, An. arabiensis, An. stephensi and An. albimanus) indicated that heterogeneity of the salivary proteome and antigenic proteins was lower among closely related anopheline species and increased with phylogenetic distance. CONCLUSION: This is the first report on the diversity of the salivary protein repertoire among species from the Anopheles genus at the protein level. This work demonstrates that a molecular diversity is exhibited among salivary proteins from closely related species despite their common pharmacological activities. The involvement of these proteins as antigenic candidates for genus-, subgenus- or species-specific immunological evaluation of individual exposure to Anopheles bites is discussed.

Multiparametric cytometry for exploration of complex cellular dynamics.

The development of polychromatic cytometry has contributed to significant progress in the field of human immunology. Although numerous functional studies of rare cell populations have been performed using this technology, here we used polychromatic cytometry to explore the dynamics of complex cellular systems implicated in innate immunity. We used PBMC stimulated with live influenza virus as an experimental model. We studied the time course of activation of PBMC, which contain DC, monocytes, and NK cells, all of which are, in addition to their innate immune properties, susceptible to Flu infection. We developed 12 color panels to investigate intracellular expression of IFN-α, TNF-α, IL-12, IL-6, IFN-γ, CD107, and influenza virus nucleoprotein simultaneously in these cell populations. These panels allowed reproducible determination of activation markers induced in DC after their direct exposure to various stimulations or in NK cells by indirect DC-mediated activation within the complex cellular environment. The ability to use a low number of cells and reduced quantities of reagents permitted us to perform kinetic experiments. The power of polychromatic cytometry associated with bioinformatic tools allowed us to analyze the multiple functional data generated as dynamic clustering maps. These maps present a readily understandable view of activation events induced in different populations of PBMC. In addition, it reveals new information on the coordination of the complex pathways induced and on the cellular interactions that sustained indirect DC-mediated NK cell activation. Our work shows that polychromatic cytometry is a tool for discoveries in unexplored complex cell systems, at the crossroads of immunology and virology. © 2012 International Society for Advancement of Cytometry.

Plasmodium falciparum infection-induced changes in erythrocyte membrane proteins.

Over the past decade, advances in proteomic and mass spectrometry techniques and the sequencing of the Plasmodium falciparum genome have led to an increasing number of studies regarding the parasite proteome. However, these studies have focused principally on parasite protein expression, neglecting parasite-induced variations in the host proteome. Here, we investigated P. falciparum-induced modifications of the infected red blood cell (iRBC) membrane proteome, taking into account both host and parasite proteome alterations. Furthermore, we also determined if some protein changes were associated with genotypically distinct P. falciparum strains. Comparison of host membrane proteomes between iRBCs and uninfected red blood cells using fluorescence-based proteomic approaches, such as 2D difference gel electrophoresis revealed that more than 100 protein spots were highly up-represented (fold change increase greater than five) following P. falciparum infection for both strains (i.e. RP8 and Institut Pasteur Pregnancy Associated Malaria). The majority of spots identified by mass spectrometry corresponded to Homo sapiens proteins. However, infection-induced changes in host proteins did not appear to affect molecules located at the outer surface of the plasma membrane. The under-representation of parasite proteins could not be attributed to deficient parasite protein expression. Thus, this study describes for the first time that considerable host protein modifications were detected following P. falciparum infection at the erythrocyte membrane level. Further analysis of infection-induced host protein modifications will improve our knowledge of malaria pathogenesis.

Exploratory Study on Application of MALDI-TOF-MS to Detect SARS-CoV-2 Infection in Human Saliva.

SARS-CoV-2 has caused a large outbreak since its emergence in December 2019. COVID-19 diagnosis became a priority so as to isolate and treat infected individuals in order to break the contamination chain. Currently, the reference test for COVID-19 diagnosis is the molecular detection (RT-qPCR) of the virus from nasopharyngeal swab (NPS) samples. Although this sensitive and specific test remains the gold standard, it has several limitations, such as the invasive collection method, the relative high cost and the duration of the test. Moreover, the material shortage to perform tests due to the discrepancy between the high demand for tests and the production capacities puts additional constraints on RT-qPCR. Here, we propose a PCR-free method for diagnosing SARS-CoV-2 based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling and machine learning (ML) models from salivary samples. Kinetic saliva samples were collected at enrollment and ten and thirty days later (D0, D10 and D30), to assess the classification performance of the ML models compared to the molecular tests performed on NPS specimens. Spectra were generated using an optimized protocol of saliva collection and successive quality control steps were developed to ensure the reliability of spectra. A total of 360 averaged spectra were included in the study. At D0, the comparison of MS spectra from SARS-CoV-2 positive patients (n = 105) with healthy healthcare controls (n = 51) revealed nine peaks that significantly distinguished the two groups. Among the five ML models tested, support vector machine with linear kernel (SVM-LK) provided the best performance on the training dataset (accuracy = 85.2%, sensitivity = 85.1%, specificity = 85.3%, F1-Score = 85.1%). The application of the SVM-LK model on independent datasets confirmed its performances with 88.9% and 80.8% of correct classification for samples collected at D0 and D30, respectively. Conversely, at D10, the proportion of correct classification had fallen to 64.3%. The analysis of saliva samples by MALDI-TOF MS and ML appears as an interesting supplementary tool for COVID-19 diagnosis, despite the mitigated results obtained for convalescent patients (D10).

Mass Cytometry Reveals Classical Monocytes, NK Cells, and ICOS+ CD4+ T Cells Associated with Pembrolizumab Efficacy in Patients with Lung Cancer.

PURPOSE: Immune checkpoint inhibitors (ICI) have revolutionized the treatment of non-small cell lung cancer (NSCLC), but predictive biomarkers of their efficacy are imperfect. The primary objective is to evaluate circulating immune predictors of pembrolizumab efficacy in patients with advanced NSCLC. EXPERIMENTAL DESIGN: We used high-dimensional mass cytometry (CyTOF) in baseline blood samples of patients with advanced NSCLC treated with pembrolizumab. CyTOF data were analyzed by machine-learning algorithms (Citrus, tSNE) and confirmed by manual gating followed by principal component analysis (between-group analysis). RESULTS: We analyzed 27 patients from the seminal KEYNOTE-001 study (median follow-up of 60.6 months). We demonstrate that blood baseline frequencies of classical monocytes, natural killer (NK) cells, and ICOS+ CD4+ T cells are significantly associated with improved objective response rates, progression-free survival, and overall survival (OS). In addition, we report that a baseline immune peripheral score combining these three populations strongly predicts pembrolizumab efficacy (OS: HR = 0.25; 95% confidence interval = 0.12-0.51; P < 0.0001). CONCLUSIONS: As this immune monitoring is easy in routine practice, we anticipate our findings may improve prediction of ICI benefit in patients with advanced NSCLC.

Optimization and Standardization of Human Saliva Collection for MALDI-TOF MS.

SARS-CoV-2 outbreak led to unprecedented innovative scientific research to preclude the virus dissemination and limit its impact on life expectancy. Waiting for the collective immunity by vaccination, mass-testing, and isolation of positive cases remain essential. The development of a diagnosis method requiring a simple and non-invasive sampling with a quick and low-cost approach is on demand. We hypothesized that the combination of saliva specimens with MALDI-TOF MS profiling analyses could be the winning duo. Before characterizing MS saliva signatures associated with SARS-CoV-2 infection, optimization and standardization of sample collection, preparation and storage up to MS analyses appeared compulsory. In this view, successive experiments were performed on saliva from healthy healthcare workers. Specimen sampling with a roll cotton of Salivette® devices appeared the most appropriate collection mode. Saliva protein precipitation with organic buffers did not improved MS spectra profiles compared to a direct loading of samples mixed with acetonitrile/formic acid buffer onto MS plate. The assessment of sample storage conditions and duration revealed that saliva should be stored on ice until MS analysis, which should occur on the day of sampling. Kinetic collection of saliva highlighted reproducibility of saliva MS profiles over four successive days and also at two-week intervals. The intra-individual stability of saliva MS profiles should be a key factor in the future investigation for biomarkers associated with SARS-CoV-2 infection. However, the singularity of MS profiles between individuals will require the development of sophisticated bio-statistical analyses such as machine learning approaches. MALDI-TOF MS profiling of saliva could be a promising PCR-free tool for SARS-CoV-2 screening.

Mouth Washing Impaired SARS-CoV-2 Detection in Saliva.

BACKGROUND: A previous study demonstrated the performance of the Salivette® (SARSTEDT, Numbrecht, Germany) as a homogeneous saliva collection system to diagnose COVID-19 by RT-qPCR, notably for symptomatic and asymptomatic patients. However, for convalescent patients, the corroboration of molecular detection of SARS-CoV-2 in paired nasopharyngeal swabs (NPS) and saliva samples was unsatisfactory. OBJECTIVES: The aim of the present work was to assess the concordance level of SARS-CoV-2 detection between paired sampling of NPSs and saliva collected with Salivette® at two time points, with ten days of interval. RESULTS: A total of 319 paired samples from 145 outpatients (OP) and 51 healthcare workers (HW) were collected. Unfortunately, at day ten, 73 individuals were lost to follow-up, explaining some kinetic missing data. Due to significant waiting rates at hospitals, most of the patients ate and/or drank while waiting for their turn. Consequently, mouth washing was systematically proposed prior to saliva collection. None of the HW were diagnosed as SARS-CoV-2 positive using NPS or saliva specimens at both time points (n = 95) by RT-qPCR. The virus was detected in 56.3% (n = 126/224) of the NPS samples from OP, but solely 26.8% (n = 60/224) of the paired saliva specimens. The detection of the internal cellular control, the human RNase P, in more than 98% of the saliva samples, underlined that the low sensitivity of saliva specimens (45.2%) for SARS-CoV-2 detection was not attributed to an improper saliva sample storing or RNA extraction. CONCLUSIONS: This work revealed that mouth washing decreased viral load of buccal cavity conducting to impairment of SARS-CoV-2 detection. Viral loads in saliva neo-produced appeared insufficient for molecular detection of SARS-CoV-2. At the time when saliva tests could be a rapid, simple and non-invasive strategy to assess large scale schoolchildren in France, the determination of the performance of saliva collection becomes imperative to standardize procedures.

Salivette, a relevant saliva sampling device for SARS-CoV-2 detection.

Background: The gold standard for COVID-19 diagnosis relies on quantitative reverse-transcriptase polymerase-chain reaction (RT-qPCR) from nasopharyngeal swab (NPS) specimens, but NPSs present several limitations. The simplicity, low invasive and possibility of self-collection of saliva imposed these specimens as a relevant alternative for SARS-CoV-2 detection. However, the discrepancy of saliva test results compared to NPSs made of its use controversial. Here, we assessed Salivettes®, as a standardized saliva collection device, and compared SARS-CoV-2 positivity on paired NPS and saliva specimens. Methods: A total of 303 individuals randomly selected among those investigated for SARS-CoV-2 were enrolled, including 30 (9.9%) patients previously positively tested using NPS (follow-up group), 90 (29.7%) mildly symptomatic and 183 (60.4%) asymptomatic. Results: The RT-qPCR revealed a positive rate of 11.6% (n = 35) and 17.2% (n = 52) for NPSs and saliva samples, respectively. The sensitivity and specificity of saliva samples were 82.9% and 91.4%, respectively, using NPS as reference. The highest proportion of discordant results concerned the follow-up group (33.3%). Although the agreement exceeded 90.0% in the symptomatic and asymptomatic groups, 17 individuals were detected positive only in saliva samples, with consistent medical arguments. Conclusion Saliva collected with Salivette® was more sensitive for detecting symptomatic and pre-symptomatic infections.

Quantification of Immune Variables from Liquid Biopsy in Breast Cancer Patients Links Vδ2+ γδ T Cell Alterations with Lymph Node Invasion.

The rationale for therapeutic targeting of Vδ2+ γδ T cells in breast cancer is strongly supported by in vitro and murine preclinical investigations, characterizing them as potent breast tumor cell killers and source of Th1-related cytokines, backing cytotoxic αß T cells. Nonetheless, insights regarding Vδ2+ γδ T cell phenotypic alterations in human breast cancers are still lacking. This paucity of information is partly due to the challenging scarcity of these cells in surgical specimens. αß T cell phenotypic alterations occurring in the tumor bed are detectable in the periphery and correlate with adverse clinical outcomes. Thus, we sought to determine through an exploratory study whether Vδ2+ γδ T cells phenotypic changes can be detected within breast cancer patients' peripheral blood, along with association with tumor progression. By using mass cytometry, we quantified 130 immune variables from untreated breast cancer patients' peripheral blood. Supervised analyses and dimensionality reduction algorithms evidenced circulating Vδ2+ γδ T cell phenotypic alterations already established at diagnosis. Foremost, terminally differentiated Vδ2+ γδ T cells displaying phenotypes of exhausted senescent T cells associated with lymph node involvement. Thereby, our results support Vδ2+ γδ T cells implication in breast cancer pathogenesis and progression, besides shedding light on liquid biopsies to monitor surrogate markers of tumor-infiltrating Vδ2+ γδ T cell antitumor activity.