Fremme 3-D Aggregering av FACS Renset Thymus- Epitelceller med EAK 16-II / EAKIIH6 Self-montering Hydrogel
Summary
This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.
Abstract
Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.
Introduction
Thymus er det primære lymfoide organ er ansvarlig for genereringen av en mangfoldig populasjon av patogen-reaktive, selv-tolerant T-celler som er avgjørende for funksjonen av ervervet immunsystemet. Det er en dynamisk organ hvor utviklings thymocytter, innvandret fra benmargen som lymfocytt progenitorceller, migrerer gjennom det svampliknende tredimensjonale (3-D) matrise av thymus-stroma, gjennomgår avstamning-spesifikasjonen og differensiering, og til slutt migrere så modne T-celler. Suksessen til dette godt programmert prosessen avhenger i stor grad av krysstale mellom de trekkende thymocytter og bolig thymus epitelceller (Tecs), den dominerende befolkningen i thymus stroma som er avgjørende for å etablere og opprettholde integriteten av thymus mikromiljøet.
Basert på deres anatomiske plassering og unik funksjon, kan Tecs deles inn i to undergrupper: den Tecs i cortex (cTECs) som er ansvarslig for valg av selv-MHC (major histocompatibility komplekse) begrensede T-celler (positiv seleksjon), og Tecs i medulla (mTECs) som er vesentlig for å eliminere autoreaktive T-celler (negativ seleksjon) 1,2. Mange faktorer (for eksempel aldring, infeksjon, bestråling, narkotika behandlinger) kan føre til irreversible skader på thymus epitel, noe som resulterer i nedsatt adaptiv immunitet. Til tross for mange forsøk, gjenopprette thymus-funksjonen har vært utfordrende på grunn av problemer med å reprodusere thymus mikromiljøet. Spesielt, er tymisk tre-dimensjonal (3-D) konfigurasjon kritisk for overlevelse og funksjon av Tecs, mens Tecs dyrket i en 2-D miljø hurtig redusere ekspresjonen av gener som kritiske for thymopoiesis 3,4.
EAK16-II (AEAEKAKAEAEAKAK) og dets C-terminale histidinylated analog EAKIIH6 (AEAEKAKAEAEAKAKHHHHHH) er lav molekylvekt, amfifile oligopeptider som er løselige i deionisert water, men gjennomgår gelering for dannelse av p-fibriller når de utsettes for ionestyrke høyere enn 20 mM NaCl (normal saltkonsentrasjon i human kroppsvæske er 154 mM). Dette miljø responsive egenskapen gjør dem allsidige byggesteiner for å danne 3D-strukturer. Den His-tag på EAKII-H6 gir en docking mekanisme, der anti-Hans IgG / Fc-bindende rekombinant protein A / G (αH6: PA / G) komplekser kan tjene som en adapter for å forankre protein narkotika og andre biomolekyler ( for eksempel antistoffer) på hydrogelen kompositt 5-8.
Vi har tidligere demonstrert at fluorescensmerkede IgG-molekyler er forankret på hydrogelen kan beholdes ved injeksjonsstedet i opp til 13 dager 9. Videre, når de αH6: pA / G adaptere forankret med anti-CD4-IgG ble tilsatt til EAK16-II / EAKIIH6 (EAK) hydrogel, CD4 + T-celler ble bestemt tatt 10. Ved hjelp av tilsvarende teknikk, har vi nylig vist at 3-D aggregering av Tecs cOuld bli fremmet i EAK hydrogel med adapter komplekser bærer TEC-spesifikk anti-EpCAM antistoffer (αH6: PA / G: αEpCAM). Når transplantert under nyre kapsler av nakne mus, kan TEC klynger innebygd i EAK hydrogel effektivt støtte utviklingen av funksjonelle T-celler in vivo 11,12.
Her viser vi vår metode for å raskt og effektivt rense Tecs med fluorescens-aktivert celle sortering (FACS) og generere 3D-TEC aggregater med EAK hydrogel system.
Protocol
Representative Results
Discussion
Mens Tecs er den dominerende befolkningen i thymus stroma og spille viktige roller for struktur og funksjon av thymus kjertler, de representerer bare ca 0,1-0,5% av den totale thymus cellularity. De er også skjøre celler som høye prosentandeler av celledød blir av og til observert følgende kollagenase fordøyelse, dvs. til behandling dissosiere Tecs fra den ekstracellulære matriks (ECM). Deres sjeldenhet (~ 200 000 per mus thymus) og skjørhet gjort det en utfordrende oppgave å isolere Tecs. Den nylige u…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Dette arbeidet ble støttet delvis av National Institutes of Health gir R21 AI113000 (WSM) og R01 AI123392 (YF).
Materials
| 1. TEC Isolation | |||
| 70% Ethanol | Decon Laboratories | 2701(1 Gallon) | Ethanol 200 Proof, deionized water |
| Dissecting scissors, straight | Fine Science Tools, Inc. | 91460-11 | |
| Graefe forceps, straight | Fine Science Tools, Inc. | 11053-10 | |
| Graefe forceps, curved | Fine Science Tools, Inc. | 11052-10 | |
| Washing solution | 1X PBS, 0.1% BSA, 2mM EDTA | ||
| 1x PBS (Phosphate buffered saline) | Gibco | 10010-023 | |
| BSA (Bovine serum albumin) | Sigma | A1470-100G | |
| EDTA (ethylenediaminetetraacetic acid) | Invitrogen | 15575-038 | |
| Digestion solution | 9 mL RPMI-1640, 0.025 mg/mL Liberase TM Research grade, 10 mM HEPES, 0.2 mg/mL DNaseI | ||
| RPMI-1640 | Gibco | 11879-020 | |
| Liberase TM Research Grade | Roche | 05 401 127 001 | referred as "purified collagenase" |
| 1M HEPES | Lonza | 17-737E | |
| Dnase I | Roche | 10 104 159 001 | |
| 50mL Centrifuge tube | Corning | 430290 | |
| 60mm tissue culture dish | Falcon | 353002 | |
| 1/2cc U-100 Insulin syringe 28G1/2 | Becton Dickinson | 329461 | |
| 5mL Polystyrene round-bottom tube | Falcon | 352058 | |
| 5ml glass pipet | Fisher Healthcare | 13-678-27E | Use for rinsing the thymic fragments. Thymic fragments tend to stick to the wall with plastic pipets. |
| MACSmix tube rotator | Miltenyi | 130-090-753 | |
| 100um Cell strainer | Falcon | 352360 | |
| Density gradient medium: OptiPrep | Axis-Shield | ||
| Name | Company | Catalog Number | Comments |
| 2. Cell sorting | |||
| 5mL Polypropylene round-bottom tube | Falcon | 352063 | |
| Anti-mouse CD16/CD32 (Fc Block) | BD Biosciences | 553142 | Use as undiluted, 2uL per sample |
| Anti-mouse CD45-PercpCy5.5 | eBioscience | 45-0451-80 | Use at 1:150, 10uL per sample |
| Anti-mouse CD326 (EpCAM)-PE | eBioscience | 12-5791-82 | Use at 1:100, 10uL per sample |
| BD Influx | BD Biosciences | ||
| Single cell analysis software | FlowJo | ||
| Name | Company | Catalog Number | Comments |
| 2. EAK gel assembly | |||
| Anti-His-Tag | AnaSpec | 29673 | "anti-His-Tag IgG" |
| Purified anti-mouse CD326 (EpCAM) | BioLegend | 118202 | "anti-EpCAM IgG" |
| Recombinant protein A/G | Pierce Biotechnology | ||
| 1.5mL Safe-Lock Tubes, Biopur, Sterile | Fisher Healthcare | 05-402-24B | referred as "1.5mL microcentrifuge tube" |
| 96-well, Tissue culture plate, Round-bottom with low evaporation lid | BD Falcon | 353917 | |
| Rocking platform: Nutator Mixer no.1105 | BD Clay Adams | ||
| 10% sucrose | Sigma | S0389 | Prepare with sterile distilled water |
| EAK16-II (AcNH-AEAEAKAKAEAEAKAK-CONH2) | American Peptide Company | custom synthesized, 10mg/mL | |
| EAKIIH6 (AcNH-AEAEAKAKAEAEAKAKHHHHHH-CONH2) | American Peptide Company | custom synthesized, 7.5mg/mL | |
| Complete medium | RPMI-1640, 10% FBS, 1% Pen/Strep, 1% L-glutamine, 1% NEAA, 5mM HEPES, 50uM 2-Mercaptoethanol | ||
| RPMI-1640 | Gibco | 11879-020 | |
| FBS (Fetal Bovine Serum) | Atlanta Biologicals | S11150 | Heat inactivated before use |
| Pen/Strep | Gibco | 15140-122 | |
| L-glutamine 200mM (100x) | Gibco | 25030-081 | |
| NEAA (non-essential amino acid) 100x | Gibco | 11140-050 | |
| 1M HEPES | BioWhittaker | 17-737E | |
| 2-Mercaptoethanol (100X) | Millipore | ES-007-E | |
| Platform shaker: The Belly Dancer | Stovall Life Sciences Inc. | model: USBDbo |
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