Title Ex vivo expansion and phenotypic characterization of mouse primary regulatory T cells
Title (croatian) Ex vivo ekspanzija i fenotipska karakterizacija mišjih primarnih T regulatornih stanica
Author Kristina Manzoni
Mentor Jelka Pohar (mentor)
Mentor Jelena Ban (komentor)
Committee member Jelka Pohar (član povjerenstva)
Committee member Nikolina Vidović (član povjerenstva)
Committee member Ivana Munitić (predsjednik povjerenstva)
Committee member Jelena Ban (član povjerenstva)
Granter University of Rijeka (Faculty of Biotechnology and Drug Development) Rijeka
Defense date and country 2024-07-09, Croatia
Scientific / art field, discipline and subdiscipline BIOTECHNICAL SCIENCES Biotechnology
Abstract Foxp3+ CD25+ regulatory T cells (Tregs) are a rare subset within the CD4+ T cell compartment, playing a vital role in immune system regulation. Because of their immunosuppressive ability, Tregs are an attractive potential new tool for treating autoimmune diseases and preventing graft rejection. Although the population of Treg cells is well-defined by the intracellular expression of the transcription factor Forkhead box protein 3 (Foxp3), no defined surface marker distinguishes Tregs from other T cells, making reliable phenotypic characterization and isolation challenging. Furthermore, most isolation and expansion protocols are designed for human Tregs, representing a significant drawback since mice are important model organisms in the preclinical phase of drug development. A range of methodologies was employed. Treg cells were isolated from mouse spleens using immunomagnetic separation. Consequently, cells were activated and expanded for 15 days, using different ratios of Treg cells-to-activation beads in the presence of mouse interleukin-2 (mIL-2) cytokine. After the staining for viability, surface, and intracellular markers, the
phenotype of Tregs and other T cell subsets was analyzed using flow cytometry (FC). To investigate the transcriptional profiles of subpopulations within the Tregs samples, we performed the 10x Genomics single-cell RNA sequencing (scRNA-seq). This study resulted in the successful establishment of isolation and expansion protocols for mouse Foxp3+ CD25+ Tregs, as well as comprehensive FC staining panels. The phenotype and transcriptome analysis revealed the heterogeneity of the Treg population already upon isolation from secondary lymphatic tissue. ScRNA-seq analysis provided a deeper insight into the Treg cells' transcriptome and revealed other important biomarkers for contaminants-free isolation and improved phenotypic characterization of Treg cells. The obtained results pave the way for developing innovative therapeutic approaches for autoimmune diseases, graft rejection and cancer, which hold a promise to greatly improve patients´quality of life.
Abstract (croatian) Foxp3+ CD25+ regulatorne T stanice (Tregs) rijetka su podskupina CD4+ T stanica, koje igraju vitalnu ulogu u regulaciji imunološkog sustava. Zbog svojih imunosupresivnih sposobnosti, Treg su zanimljive kao potencijalno novo sredstvo za liječenje autoimunih bolesti i sprječavanje odbacivanja presatka. Iako je populacija Treg stanica dobro definirana unutarstaničnom ekspresijom transkripcijskog faktora Foxp3, ne postoji definirani površinski marker koji bi razlikovao Tregs od ostalih T stanica, što pouzdanu fenotipsku karakterizaciju i izolaciju čini izazovnom. Nadalje, većina protokola izolacije i ekspanzije dizajnirana je za ljudske Treg stanice, što predstavlja značajan nedostatak jer su miševi važni modelni organizmi u pretkliničkoj fazi razvoja lijeka. Primijenjen je niz metodologija. Treg stanice izolirane su iz slezena miševa pomoću imunomagnetske separacije. Stanice su zatim aktivirane i ekspandirane tijekom 15 dana, korištenjem različitih omjera Treg stanica i aktivacijskih kuglica u prisutnosti interleukin-2 citokina. Nakon bojanja antitijelima za vijabilnost, površinske i unutarstanične markere, fenotip Treg stanica i drugih podskupova T stanica analiziran je protočnom citometrijom. Kako bismo istražili transkripcijske profile subpopulacija unutar Treg uzoraka, izvršili smo 10x Genomics single-cell RNA sekvenciranje (scRNAseq). Ova studija rezultirala je uspješnom uspostavom protokola izolacije i ekspanzije za mišje Foxp3+ CD25+ Tregs, kao i definiranim sveobuhvatnim panelima kombinacije antitijela za protočnu citometriju. Analiza fenotipa i transkriptoma otkrila je heterogenost Treg populacije već pri samoj izolaciji iz sekundarnog limfnog tkiva.
ScRNA-seq analiza pružila je dublji uvid u transkriptom Treg stanica i otkrila druge važne biomarkere za izolaciju bez kontaminanata i poboljšanu fenotipsku karakterizaciju Treg stanica. Dobiveni rezultati otvaraju put prema razvoju inovativnih pristupa liječenju za autoimune bolesti, odbacivanje presatka i rak, koji obećavaju značajno poboljšanje kvalitete života pacijenata.
Keywords
regulatory T cells
mice
protocol optimization
flow cytometry
single-cell RNA sequencing
Keywords (croatian)
regulatorne T stanice
miševi
optimizacija protokola
protočna citometrija
single-cell RNA sekvenciranje
Language english
URN:NBN urn:nbn:hr:193:805678
Project Number: MN-0013-105 Title: Story of regulatory T cells told through their transcriptome Acronym: TREXCell Leader: Jelka Pohar Jurisdiction: Slovenia Funder: ARIS
Project Number: J3-3084 Title: Inducible Programming of CAR T Cell Intrinsic Properties for Cancer Immunotherapy Leader: Anže Smole Jurisdiction: Slovenia Funder: ARIS
Project Number: L4-3181 Title: Hierarchical DNA assembly for advanced applications in biopharmaceuticals production and cell therapy Leader: Marko Dolinar Jurisdiction: Slovenia Funder: ARIS
Project Number: J3-4516 Title: Neoantigens in non small cell lung cancer Leader: Julij Šelb Jurisdiction: Slovenia Funder: ARIS
Project Number: P1-0245 Title: Ecotoxicology, toxicogenomics and carcinogenesis Leader: Bojana Žegura Jurisdiction: Slovenia Funder: ARIS
Project Number: 10ICIGEN Title: Immunology and Cellular Immunotherapy Lab Leader: Jelka Pohar, Anže Smole Jurisdiction: Slovenia Funder: National Institute of Biology
Project Number: InTREGing Title: Vpogled v transkriptom regulatornih celic T (Treg) v ključnih stopnjah imunskega odziva Leader: Jelka Pohar Jurisdiction: Slovenia Funder: National Institute of Biology
Project Number: 10X Challenge Title: Single cell profiling of regulatory T cell subsets Leader: Jelka Pohar Jurisdiction: Slovenia Funder: Labena d.o.o.
Study programme Title: Biotechnology in medicine Study programme type: university Study level: graduate Academic / professional title: magistar/magistra biotehnologije u medicini (magistar/magistra biotehnologije u medicini)
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Created on 2024-07-18 07:42:36