Nye værktøjer til at udvide regulatoriske T celler fra HIV-1-inficerede personer
Summary
CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.
Abstract
CD4 + Regulatoriske T-celler (tregs) er potente immunmodulatorer og tjene en vigtig funktion i human immun homøostase. Udtømning af tregs har ført til målelige stigninger i antigen-specifikke T-celle responser i vaccine indstillinger for cancer og infektiøse patogener. Men deres rolle i HIV-1 immuno-patogenese er stadig kontroversielt, da de enten kunne tjene til at undertrykke skadelige HIV-1-associerede immun aktivering og dermed bremse HIV-1 sygdomsprogression eller alternativt undertrykke HIV-1-specifik immunitet og dermed fremme virus spredes. Forståelse og modulerende Treg funktion i forbindelse med HIV-1 kan føre til potentielle nye strategier for immunterapi eller HIV-vacciner. Men vigtige åbne spørgsmål forblive på deres rolle i forbindelse med HIV-1 infektion, som skal undersøges nøje.
Udgør omtrent 5% af humane CD4 + T-celler i det perifere blod, studere Treg befolkning har vist sig at være vanskeligt, especially i HIV-1-inficerede personer, hvor HIV-1-associerede CD4 T-celle og med det Treg udtynding opstår. Karakteriseringen af regulatoriske T-celler i individer med fremskreden HIV-1 sygdom eller vævsprøver, som kun meget små biologiske prøver kan opnås, er derfor yderst udfordrende. Vi foreslår en teknisk løsning til at overvinde disse begrænsninger ved hjælp isolation og udvidelse af tregs fra HIV-1-positive individer.
Her beskriver vi en let og robust metode til med succes at udvide tregs isoleret fra HIV-1-inficerede individer in vitro. Flow-sorteret CD3 + CD4 + CD25 + CD127 low tregs blev stimuleret med anti-CD3/anti-CD28 overtrukne perler og dyrket i nærværelse af IL-2. Det ekspanderede tregs udtrykte høje niveauer af foxp3, CTLA4 og HELIOS forhold til konventionelle T-celler og blev vist at være yderst undertrykkende. Lettere adgang til et stort antal tregs vil give forskere til at løse important spørgsmål vedrørende deres rolle i HIV-1 immunopatogenese. Vi tror at besvare disse spørgsmål kan give nyttig indsigt til udviklingen af en effektiv HIV-1-vaccine.
Introduction
Med mere end 34 millioner mennesker lever med hiv / aids på verdensplan, og en anslået 2,5 millioner mennesker smittet i 2011, er behovet for en effektiv HIV-vaccine for at bremse den globale hiv-epidemi er altoverskyggende. Men på trods af tre årtiers intense forskningsindsats, har HIV-1-vaccine effektundersøgelser til dato resulteret i kun en beskeden beskyttelse 1-3 og korrelater for beskyttende immunitet forbliver dårligt forstået. Belyse naturen af immunresponset nødvendig for beskyttelse er afgørende for strategisk udformning af en effektiv HIV-1-vaccine og andre immunterapeutiske strategier rettet mod HIV-1 infektion.
Naturlige CD4 + regulatoriske T-celler (tregs) er afgørende for opretholdelsen af immun cellehomeostase ved at kontrollere overdreven immun aktivering, hvilket begrænser immunmedieret vævsskade. Men de kan også undertrykke immunresponser mod patogener og forhindre deres clearance. Kræft og HEPAtitis B vaccine studier har vist, at reducere aktiviteten af tregs kan forbedre vaccine respons og antigenspecifik immunitet mod virus 4-7. Men i forbindelse med HIV-1 infektion forbliver nøjagtige virkning af regulatoriske T-celler ufuldstændigt forstået. Tregs blev vist at falde virusreplikation i aktiverede T-celler 8 og muligvis påvirke immun aktivering 9. De blev også vist sig at undertrykke HIV-1-specifikke immunreaktioner, hvilket kan få negative resultater for sygdomsprogression 10,11. Således, før at være i stand til at modulere Treg aktivitet at forbedre effektiviteten af en HIV-1-vaccine, er det vigtigt at opnå yderligere indsigt i deres funktion i denne sygdom sammenhæng.
Humane CD4 +-regulatoriske T-celler er en relativt begrænset cellepopulation, hvilket svarer til omkring 5% af CD4 + T-celler i det perifere blod, og deres absolutte tal yderligere fald med HIV-associerede CD4 + T-celle depletion 12 </ Sup>. Aktuelle analyser at vurdere Treg funktion, såsom T-celleproliferationsassays med Treg co-kultur, brug relativt store celle tal 12.. Derfor karakteriserer funktion og specificitet af regulatoriske T-celler hos personer med fremskreden HIV-1 sygdommen har været en udfordring, på trods af deres betydning for HIV patogenese.
Ex vivo isolation og udvidelse af tregs fra HIV-1 patienter kunne udgøre en løsning til at overvinde nogle af disse begrænsninger. Her beskriver vi en let og robust protokol for at udvide funktionel tregs afledt fra HIV-1-inficerede individer in vitro, vi yderligere forklare, hvordan man fænotype dem og teste deres undertrykkende funktion ved hjælp flowcytometriske assays. Vi tror, at dette protokol vil lette adgangen til tregs og hjælp til at forstå deres rolle i HIV-1 sygdomsprogression.
Protocol
Representative Results
Discussion
Using the protocol described above, Tregs can be successfully isolated and expanded from HIV-1-infected individuals in vitro. Expanded Tregs express high levels of FOXP3, CTLA4 and HELIOS, are highly suppressive and display a highly demethylated Treg-Specific Demethylation Region (TSDR) locus of the FOXP3 gene (data not shown) 15, suggesting true origin from the regulatory T cell lineage, as opposed to activation-induced transient FOXP3 upregulation. Deep sequencing demonstrated that the TCR repertoir…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported in part by research funding from the Elisabeth Glaser Pediatric AIDS Foundation (Pediatric HIV Vaccine Program Award MV-00-9-900-1429-0-00 to MMA), MGH/ECOR (Physician Scientist Development Award to MMA), NIH NIAID (KO8219 AI074405 and AI074405-03S1 to MMA), and the Harvard University Center for AIDS Research (CFAR), an NIH funded program (P30 AI060354) which is supported by the following NIH Co-Funding and Participating Institutes and Centers: NIAID, NCI, NICHD, NHLBI, NIDA, NIMH, NIA, FIC, and OAR. These studies were furthermore supported by the Bill & Melinda Gates Foundation and the Terry and Susan Ragon Foundation.
Materials
| Reagents | |||
| RosetteSep Human CD4+ T Cell Enrichment Cocktail | Stemcells technologies | 15062 | |
| PBS | Sigma | D8537 | |
| FBS | Sigma | F4135 | |
| Histopaque | Sigma | H8889 | |
| Anti-CD3-PECy7 | BD Pharmingen | 557851 | |
| Anti-CD4-FITC | eBioscience | 11-0049-42 | |
| Anti-CD25-APC | eBioscience | 17-0259-42 | |
| Anti-CD127-PE | BD Pharmingen | 557938 | |
| Round-Bottom tube with 35 μm a nylon mesh | BD Falcon | 352235 | |
| X-VIVO 15 | Lonza | 04-418Q | |
| Penicillin/Streptomycin | Mediatech | 30-001-Cl | |
| Human Serum | Gemini Bio-Products | 100-512 | |
| Human T-activator CD3/CD28 | Life Technologies | 111.31D | |
| IL-2 | NIH Aids Research & Reference Reagent Program | 136 | |
| LIVE/DEAD Fixable Violet Dead Cell Stain Kit | Life technologies | L34955 | |
| Anti-CD4-qdot-655 | Life Technologies | Q10007 | |
| Anti-CD25-PECy5 | eBiosciences | 15-0259-42 | |
| Foxp3 / Transcription Factor Staining Buffer Set | eBiosciences | 00-5523-00 | |
| Anti-FOXP3-PE | eBiosciences | 12-4776-42 | |
| Anti-HELIOS-FITC | Biolegend | 137204 | |
| Anti-CTLA4-APC | BD Pharmingen | 555855 | |
| CellTrace Violet Cell Proliferation Kit | Life Technologies | C34557 | |
| Vybrant CFDA SE Cell Tracer Kit | Life Technologies | V12883 | |
| HEPES | Mediatech | 25-060-Cl | |
| Treg Suppression inspector | Miltenyi Biotec | 130-092-909 | |
| Anti-CD4-APC | BD Pharmingen | 340443 | |
| Anti-CD8-AF700 | BD Pharmingen | 557945 | |
| RPMI 1640 | Sigma | R0883 | |
| Glutamine | Mediatech | 25-002-Cl | |
| Materials | |||
| BD Vacutainer Blood Collection Tube w/ ACID CITRATE DEXTROSE (ACD) | Becton, Dickinson and Company (BD) | 364606 | |
| FACSAria IIu Cell Sorter | BD Biosciences | – | |
| LSR II Flow Cytometer | BD Biosciences | – | |
| FlowJo | Tree Star | v887 |
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