flow-cytometry-fluorescence-activated-cell-sorting-facs-isolation

Flow Cytometry and Fluorescence-Activated Cell Sorting (FACS): Isolation of Splenic B Lymphocytes

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3
1 Unit for Lymphopoiesis, Department of Immunology, Pasteur ...

magnetic-activated-cell-sorting-macs-isolation-of-thymic-t-lymphocytes

Magnetic Activated Cell Sorting (MACS): Isolation of Thymic T Lymphocytes

Source: Meunier Sylvain1,2,3, Perchet Thibaut1,2,3, Sophie Novault4, Rachel Golub1,2,3
1 Unit for Lymphopoiesis, Department of Immunology, Pasteur ...

cell-cycle-analysis-assessing-cd4-cd8-t-cell-proliferation-after

Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation After Stimulation Using CFSE Staining and Flow Cytometry

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3
1 Unit for Lymphpoiesis, Department of Immunology, Pasteur ...

adoptive-cell-transfer-introducing-donor-mouse-splenocytes-to-host

Adoptive Cell Transfer: Introducing Donor Mouse Splenocytes to a Host Mouse and Assessing Success via FACS

analysis-cell-suspensions-isolated-from-solid-tissues-spectral-flow

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

isolation-characterization-immune-cells-from-micro-dissected-mouse

Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can ...

high-quality-brain-bone-marrow-nuclei-preparation-for-single-nuclei

High-Quality Brain and Bone Marrow Nuclei Preparation for Single Nuclei Multiome Assays

Single-cell analysis has become the approach of choice for unraveling the complexity of biological processes that require assessing the variability of ...

Pasteur Institute

Institut Pasteur

Institut Pasteur, Université Paris Cité

Ideal vaccines should be stable, safe, molecularly defined, and out-of-shelf reagents efficient at triggering effector and memory Ag-specific T cell-based immune responses. Dendritic cell-derived exosomes could be considered as novel peptide-based vaccines because exosomes harbor a discrete set of proteins, bear functional MHC class I and II molecules that can be loaded with synthetic peptides of choice, and are stable reagents that were safely used in pioneering phase I studies. However, we showed in part I that exosomes are efficient to promote primary MHC class I-restricted effector CD8(+) T cell responses only when transferred onto mature DC in vivo. In this work, we bring evidence that among the clinically available reagents, Toll-like receptor 3 and 9 ligands are elective adjuvants capable of triggering efficient MHC-restricted CD8(+) T cell responses when combined to exosomes. Exosome immunogenicity across species allowed to verify the efficacy of good manufactory procedures-manufactured human exosomes admixed with CpG oligonucleotides in prophylactic and therapeutic settings of melanoma in HLA-A2 transgenic mice. CpG adjuvants appear to be ideal adjuvants for exosome-based cancer vaccines.

Exosomes as potent cell-free peptide-based vaccine. II. Exosomes in CpG adjuvants efficiently prime naive Tc1 lymphocytes leading to tumor rejection.

BACKGROUND: DC derived-exosomes are nanomeric vesicles harboring functional MHC/peptide complexes capable of promoting T cell immune responses and tumor rejection. Here we report the feasability and safety of the first Phase I clinical trial using autologous exosomes pulsed with MAGE 3 peptides for the immunization of stage III/IV melanoma patients. Secondary endpoints were the monitoring of T cell responses and the clinical outcome. PATIENTS AND METHODS: Exosomes were purified from day 7 autologous monocyte derived-DC cultures. Fifteen patients fullfilling the inclusion criteria (stage IIIB and IV, HLA-A1+, or -B35+ and HLA-DPO4+ leukocyte phenotype, tumor expressing MAGE3 antigen) were enrolled from 2000 to 2002 and received four exosome vaccinations. Two dose levels of either MHC class II molecules (0.13 versus 0.40 x 1014 molecules) or peptides (10 versus 100 mug/ml) were tested. Evaluations were performed before and 2 weeks after immunization. A continuation treatment was performed in 4 cases of non progression. RESULTS: The GMP process allowed to harvest about 5 x 1014 exosomal MHC class II molecules allowing inclusion of all 15 patients. There was no grade II toxicity and the maximal tolerated dose was not achieved. One patient exhibited a partial response according to the RECIST criteria. This HLA-B35+/A2+ patient vaccinated with A1/B35 defined CTL epitopes developed halo of depigmentation around naevi, a MART1-specific HLA-A2 restricted T cell response in the tumor bed associated with progressive loss of HLA-A2 and HLA-BC molecules on tumor cells during therapy with exosomes. In addition, one minor, two stable and one mixed responses were observed in skin and lymph node sites. MAGE3 specific CD4+ and CD8+ T cell responses could not be detected in peripheral blood. CONCLUSION: The first exosome Phase I trial highlighted the feasibility of large scale exosome production and the safety of exosome administration.

Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of thefirst phase I clinical trial.

Dendritic-cell-derived exosomes (DEX) secreted after dendritic cell loading with tumor peptides were found to mediate tumor rejection in mice. This observation prompted us to demonstrate that MHC class I/peptide complexes harbored onto exosomal membranes were capable of priming cytotoxic T cells and to mediate rejection of tumors expressing the relevant antigens. Moreover, DEX also promote NK cell activation in immunocompetent mice and NK cell-dependent antitumor effects. The first Phase I trial using DEX to immunize melanoma patients revealed the feasibility of DEX production in stage IV melanoma, their safety in long-term follow up and their bioactivity in vivo.

The potential of exosomes in immunotherapy of cancer.

Tumor growth promotes the expansion of CD4+CD25+ regulatory T (T reg) cells that counteract T cell-mediated immune responses. An inverse correlation between natural killer (NK) cell activation and T reg cell expansion in tumor-bearing patients, shown here, prompted us to address the role of T reg cells in controlling innate antitumor immunity. Our experiments indicate that human T reg cells expressed membrane-bound transforming growth factor (TGF)-beta, which directly inhibited NK cell effector functions and down-regulated NKG2D receptors on the NK cell surface. Adoptive transfer of wild-type T reg cells but not TGF-beta-/- T reg cells into nude mice suppressed NK cell-mediated cytotoxicity, reduced NKG2D receptor expression, and accelerated the growth of tumors that are normally controlled by NK cells. Conversely, the depletion of mouse T reg cells exacerbated NK cell proliferation and cytotoxicity in vivo. Human NK cell-mediated tumor recognition could also be restored by depletion of T reg cells from tumor-infiltrating lymphocytes. These findings support a role for T reg cells in blunting the NK cell arm of the innate immune system.

CD4+CD25+ regulatory T cells inhibit natural killer cell functions in a transforming growth factor-beta-dependent manner.

Dendritic cell-derived exosomes (DEX) are nanomeric vesicles harboring MHC/peptide complexes capable of promoting primary T cell responses and tumor rejection in the presence of adjuvants. In this study, we show that, in the absence of adjuvants, DEX mediate potent Ag-dependent antitumor effects against preestablished tumors in mice pretreated with immunopotentiating dosing of cyclophosphamide. Cyclophosphamide could 1) abolish the suppressive function of CD4+CD25+Foxp3+ regulatory T cells, 2) markedly enhance the magnitude of secondary but not primary CTL responses induced by DEX vaccines, 3) synergize with DEX in therapy but not prophylaxis tumor models. Therefore, therapeutic vaccines such as DEX aimed at boosting tumor-primed effector T cells could benefit procedures that minimize the effects of CD4+CD25+ regulatory T cells.

Chemoimmunotherapy of tumors: cyclophosphamide synergizes with exosome based vaccines.

Dendritic cell (DC) derived-exosomes (Dex) are nanomeric vesicles harboring functional MHC/peptide complexes promoting T cell-dependent tumor rejection. In the first Phase I trial using peptide-pulsed Dex, the observation of clinical regressions in the absence of T cell responses prompted the search for alternate effector mechanisms. Mouse studies unraveled the bioactivity of Dex on NK cells. Indeed, Dex promoted an IL-15Ralpha- and NKG2D-dependent NK cell proliferation and activation respectively, resulting in anti-metastatic effects mediated by NK1.1(+) cells. In humans, Dex express functional IL-15Ralpha which allow proliferation and IFNgamma secretion by NK cells. In contrast to immature DC, human Dex harbor NKG2D ligands on their surface leading to a direct engagement of NKG2D and NK cell activation ex vivo. In our phase I clinical trial, we highlight the capacity of Dex based-vaccines to restore the number and NKG2D-dependent function of NK cells in 7/14 patients. Altogether, these data provide a mechanistic explanation on how Dex may stimulate non MHC restricted-anti-tumor effectors and induce tumor regression in vivo.

Dendritic cell-derived exosomes promote natural killer cell activation and proliferation: a role for NKG2D ligands and IL-15Ralpha.

Flow cytometry, initially developed to analyze surface protein expression in hematopoietic cells, has increased in analytical complexity and is now widely used to identify cells from different tissues and organisms. As a consequence, data analysis became increasingly difficult due the need of large multi-parametric compensation matrices and to the eventual auto-fluorescence frequently found in cell suspensions obtained from solid organs. In contrast with conventional flow cytometry that detects the emission peak of fluorochromes, spectral flow cytometry distinguishes the shapes of emission spectra along a large range of continuous wave lengths. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable to discriminate fluorochromes with similar emission peaks and provide multi-parametric analysis without compensation requirements. Here we show that spectral flow cytometry achieves a 21-parametric (19 fluorescent probes) characterization and deals with auto-fluorescent cells, providing high resolution of specifically fluorescence-labeled populations. Our results showed that spectral flow cytometry has advantages in the analysis of cell populations of tissues difficult to characterize in conventional flow cytometry, such as heart and intestine. Spectral flow cytometry thus combines the multi-parametric analytical capacity of the highest performing conventional flow cytometry without the requirement for compensation and enabling auto-fluorescence management.

Spectral Cytometry Has Unique Properties Allowing Multicolor Analysis of Cell Suspensions Isolated from Solid Tissues.

The emergence and diversification of cell types is a leading factor in animal evolution. So far, systematic characterization of the gene regulatory programs associated with cell type specificity was limited to few cell types and few species. Here, we perform whole-organism single-cell transcriptomics to map adult and larval cell types in the cnidarian Nematostella vectensis, a non-bilaterian animal with complex tissue-level body-plan organization. We uncover eight broad cell classes in Nematostella, including neurons, cnidocytes, and digestive cells. Each class comprises different subtypes defined by the expression of multiple specific markers. In particular, we characterize a surprisingly diverse repertoire of neurons, which comparative analysis suggests are the result of lineage-specific diversification. By integrating transcription factor expression, chromatin profiling, and sequence motif analysis, we identify the regulatory codes that underlie Nematostella cell-specific expression. Our study reveals cnidarian cell type complexity and provides insights into the evolution of animal cell-specific genomic regulation.

Cnidarian Cell Type Diversity and Regulation Revealed by Whole-Organism Single-Cell RNA-Seq.

The HIV-1 Env protein is exposed at the surface of virions and infected cells. Env fluctuates between different closed and open structural states and these conformations influence both viral infectivity and sensitivity to antibody binding and neutralization. We established a flow virometry assay to visualize Env proteins at the surface of human immunodeficiency virus type 1 (HIV-1) virions. The assay is performed on ultracentrifuged fluorescent viral particles that are stained with a panel of broadly neutralizing antibodies (bNAbs) and nonneutralizing antibodies (nnAbs) that probe different epitopes of Env. We used this assay to compare Env at the surface of producer cells and viral particles and to analyze the effect of Nef, CD4, and SERINC5 on Env accessibility to antibodies. We studied the laboratory-adapted strain NL4-3 and two transmitted/founder viruses, THRO and CH058. We confirm that antibody accessibility varies between viral strains and show that Nef, CD4, and SERINC5 additively impact Env conformations. We further demonstrate that the Env accessibility profile on virions is globally similar to that observed on HIV-1-infected cells, with some noticeable differences. For instance, nnAbs bind to virions more efficiently than to producer cells, likely reflecting changes in Env conformational states on mature viral particles. This test complements other techniques and provides a convenient and simple tool for quantifying and probing the structure of Env at the virion surface and to analyze the impact of viral and cellular proteins on these parameters. HIV-1 Env conformation is one of the key parameters determining viral infectivity. The flow virometry-based assay developed in this study allows for the characterization of proteins incorporated in HIV-1 particles. We studied the conformation of HIV-1 Env and the impact that the viral protein Nef and the cellular proteins CD4 and SERINC5 have on Env accessibility to antibodies. Our assay permitted us to highlight some noticeable differences in the conformation of Env between producer cells and viral particles. It contributes to a better understanding of the actual composition of HIV-1 particles.

Flow Cytometry Analysis of HIV-1 Env Conformations at the Surface of Infected Cells and Virions: Role of Nef, CD4, and SERINC5.

Whereas extracellular vesicle (EV) research has become commonplace in different biomedical fields, this field of research is still in its infancy in mycology. Here we provide a robust set of data regarding the structural and compositional aspects of EVs isolated from the fungal pathogenic species  and . Using cutting-edge methodological approaches including cryogenic electron microscopy and cryogenic electron tomography, proteomics, and flow cytometry, we revisited cryptococcal EV features and suggest a new EV structural model, in which the vesicular lipid bilayer is covered by mannoprotein-based fibrillar decoration, bearing the capsule polysaccharide as its outer layer. About 10% of the EV population is devoid of fibrillar decoration, adding another aspect to EV diversity. By analysing EV protein cargo from the three species, we characterized the typical  EV proteome. It contains several membrane-bound protein families, including some Tsh proteins bearing a SUR7/PalI motif. The presence of known protective antigens on the surface of  EVs, resembling the morphology of encapsulated virus structures, suggested their potential as a vaccine. Indeed, mice immunized with EVs obtained from an acapsular  mutant strain rendered a strong antibody response in mice and significantly prolonged their survival upon  infection.

ArticleTotal : 7	Year
Publication title	2023
Publication title	2023
Publication title	2023
Publication title	2023
Publication title Cited by 4	2017

Article	Year
Exosomes as potent cell-free peptide-based vaccine. II. Exosomes in CpG adjuvants efficiently prime naive Tc1 lymphocytes leading to tumor rejection. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 14764679	2004
Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of thefirst phase I clinical trial. Journal of translational medicine\| PubMed ID: 15740633	2005
The potential of exosomes in immunotherapy of cancer. Blood cells, molecules & diseases\| PubMed ID: 16027014	2005
CD4+CD25+ regulatory T cells inhibit natural killer cell functions in a transforming growth factor-beta-dependent manner. The Journal of experimental medicine\| PubMed ID: 16230475	2005
Chemoimmunotherapy of tumors: cyclophosphamide synergizes with exosome based vaccines. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 16493027	2006
Dendritic cell-derived exosomes promote natural killer cell activation and proliferation: a role for NKG2D ligands and IL-15Ralpha. PloS one\| PubMed ID: 19319200	2009
Spectral Cytometry Has Unique Properties Allowing Multicolor Analysis of Cell Suspensions Isolated from Solid Tissues. PloS one\| PubMed ID: 27500930	2016
Cnidarian Cell Type Diversity and Regulation Revealed by Whole-Organism Single-Cell RNA-Seq. Cell\| PubMed ID: 29856957	2018
Flow Cytometry Analysis of HIV-1 Env Conformations at the Surface of Infected Cells and Virions: Role of Nef, CD4, and SERINC5. Journal of virology\| PubMed ID: 31852789	2020
extracellular vesicles properties and their use as vaccine platforms. Journal of extracellular vesicles\| PubMed ID: 34377375	2021

Sophie Novault

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