Mange eksperimentelle systemer er blevet anvendt til at forstå de mekanismer, der regulerer T celle udvikling og funktion i en immunreaktion. Her en genetisk tilgang med retroviral transduktion beskrives, som er økonomisk, tid effektivt, og vigtigst, meget informative identificere regulatoriske pathways.
Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.
The immune response must be highly regulated to eliminate infections but prevent attacks on self-tissue that lead to autoimmunity. Helper T cells play an essential role in regulating the immune response, and a great deal of effort has been undertaken to understand their development and function (illustrated in several recent reviews 1-3). However, many questions remain, and many approaches have been utilized to study the mechanisms controlling helper T cell development and function. These have ranged from the use of in vitro cell culture systems to whole animals. Cell culture systems, especially those using cell lines, offer the benefit of ease of use and the ability to generate large amount of material to do sophisticated biochemical analyses. However, they suffer from their limited ability to reproduce the actual conditions occurring in an immune response. In contrast, whole animal experiments offer the benefit of relevance, but they can suffer from difficulties in manipulation and the ability to perform precise controls in addition to their large costs and ethical implications. Nevertheless, the vast majority of helper T cells studies today still require the use of whole animal experiments involving primary T cells because of the inability of cell lines to duplicate the exact steps occurring in the whole animal. Therefore, it is essential to utilize cost effective approaches that are highly informative.
Genetics is one powerful tool to study helper T cell development and function, yet traditional methods involving gene knockouts or transgenes are time consuming and expensive so they are often out of reach of small labs. However, retroviral transduction offers a powerful, rapid and, cost effective genetic approach to study the mechanisms of specific gene products. Therefore, it is commonly used in papers studying helper T cell development and function.
We have optimized a procedure for retroviral transduction of helper T cells. It utilizes the pMIG (Murine stem cell virus-Internal ribosomal entry site-Green fluorescent protein) retroviral expression vector, in which the gene of interest can be cloned and thereby expressed from the retrovirus long terminal repeat (LTR) 4. In addition, downstream of the inserted gene of interest is an internal ribosome entry sequence (IRES) followed by the green fluorescent protein (GFP) gene so transduced cells can easily be followed by their expression of GFP. The vector was originally derived from the Murine Stem Cell Virus (MSCV) vectors, which contain mutations in repressor binding sites in the LTRs making them resistant to silencing and thus, giving high expression in many cell types including helper T cells 5,6. Production of high titer retrovirus requires a simple transient transfection protocol of human embryonic kidney (HEK) 293T cells with the MIG vector and a helper virus vector that expresses the retroviral GAG, Pol, and Env genes. For this the pCL-Eco helper virus vector 7 works well in producing high titer replication incompetent retroviruses.
Here these protocols for retroviral production and transduction of primary murine T cells are described in addition to some of our results using this approach to study miRNA regulation of gene expression controlling helper T cell differentiation. miRNAs are small RNAs of approximately 22 nucleotides in length that post-transcriptionally regulate gene expression by targeting homologous sequences in protein encoding messenger RNAs and suppressing translation and inducing message instability 8,9. miRNAs play critical roles in developmental gene regulation. They are essential in the earliest stages of development, as embryos that cannot produce miRNAs die at a very early stage 10. In addition miRNAs are important later on in the development of many tissues. They are thought to function by fine-tuning the expression of genes required for developmental programs 1. In helper T cells miRNAs play multiple roles and are required for regulatory T cell (Treg) development 11-14. We used retroviral transduction as a means to dissect the mechanisms of miRNA regulation of Treg differentiation 15. Through such studies important individual miRNAs were determined by retroviral-mediated overexpression. Subsequently, relevant genes regulated by these miRNAs were identified in order to understand the molecular pathways regulated by miRNAs in helper T cell differentiation.
Retroviral medieret overekspression af gener er en effektiv måde at analysere funktionen i hjælper-T-celler, som deres udvikling og funktion ofte er bestemt af ekspressionsniveauet af nøgleregulatorer. Imidlertid er forsigtig fortolkning af resultaterne påkrævet, fordi udtryk niveauer langt over den endogene gen kan introducere mange artefakter. Derfor bør denne teknik kombineres med andre for at verificere relevansen af funktion. For eksempel bør overekspression suppleres med reduceret udtryk ved hjælp siRNA'er eller gen-knockout, hvis tilgængelig. Med miRNA, vi suppleret overekspression eksperimenter med dem for blokering ved hjælp af virus, der overudtrykt kunstig miRNA rettet mod websteder, der fungerede som kompetitive inhibitorer for en miRNA 15. Retrovirale transducerede celler kan også anvendes i biokemiske assays involverer RNA og protein analyse. Men en væsentlig begrænsning af disse forsøg er effektiviteten af transduktion resulting i en blandet population af transducerede og utransducerede celler. Derfor vil disse assays sandsynligvis kræve sortering af GFP + populationen. Endelig bør in vitro-differentieringsfaktorer assays kombineres med in vivo-forsøg, og en måde, hvorpå dette kan opnås, er ved adoptivt at overføre de transducerede T-celler i mus og efter deres differentiering og deres virkning på immunresponset.
En af de vigtigste begrænsninger for dette system er størrelsen af RNA-genomet, som kan pakkes ind i retrovirale capsid. Det er vores erfaring, den maksimale indsats størrelse for MIG retroviral system, der giver god virus produktion er 3-3,5 kb. Derfor kan større gener ikke analyseres med dette system, da de giver dårlige virustitere. Men de fleste gener er mindre end denne størrelse, så dette system er anvendeligt til en lang række af gen undersøgelser.
Med retroviral transduktion, flere alternativer inden for disse protocols er blevet anvendt. Mange forskere har anvendt emballage cellelinier, som stabilt udtrykker de retrovirale gener (f.eks referere 16). Vi har imidlertid opnået de højeste titere under anvendelse af standard HEK 293T-celler med co-transfektion af PCL-Eco hjælpevirus-vektor. Isolering af naive T-hjælperceller kan også opnås gennem cellesortering snarere end den magnetisk perle og celleseparation kolonne protokol, men dette kræver adgang til en cellesorteringsapparat, og omkostningerne for sortering tid er typisk højere end de perle reagenser. Endelig er der variationer på aktivering betingelser, der anvendes til at differentiere hjælper T-celler i de forskellige undergrupper. For eksempel TCR-stimulering af celler til for længe, før udsættelse for Treg inducerende betingelser kan hæmme deres induktion 16. Dette kan være et problem, fordi retroviral ekspression kræver celledeling induceret af stimulering af celler. Ikke desto mindre har vi fundet en effektiv treg induktion ved hjælp af denne protokol med O / N activation før retroviral transduktion.
Inden for disse protokoller, vellykket applikation kræver flere faktorer. Høj titer retrovirus præparater brug effektiv transfektion af HEK 293T-celler så høj kvalitet DNA og præcist rede 2x HBS er vigtige. Desuden celledensiteten af HEK 293T-celler skal være omkring 50% på punktet af transfektion, fordi god ekspression af det transficerede DNA kræver, at cellerne er aktivt voksende, og dette vil blive inhiberet, hvis cellerne er for sparsomme eller tæt. Celler ved den optimale tæthed under transfektion bør nå konfluens på et tidspunkt under trin af virus indsamling, men de vil fortsætte med at producere høj titer virusstammer hele vejen igennem til den sidste samling. Effektiv differentiering af hjælper T-celler kræver god cellernes kvalitet så sikre, at isolerede celler er renheden illustreret i figur 1. Ligeledes kvaliteten af cellerne er afhængig af mus frabout som de blev isoleret. Til disse undersøgelser har vi brugt 6-8 uger gamle C57BL / 6-mus. Ældre mus kan have mindre naive celler, og andre stammer kan variere i deres differentiering. For eksempel BALB / c-mus er mere tilbøjelige til Th2-reaktioner end C57BL / 6-mus 17, således som anført ovenfor, C57BL / 6 T-celler kan være svært at inducere et Th2-respons. Desuden kan enhver af de differentieringstilstande variere lidt fra laboratorium til laboratorium, og effekten af gen overekspression må kun blive synlige i sub-optimale betingelser, så cytokin koncentrationer i de forskellige polarisering betingelser kan være nødvendigt at titreres. Endelig kan virkningerne af den overudtrykte gen eller polarisering betingelser på celleproliferation påvirke transduktion effektivitet, så måler effekt af genet af interesse kan kræve optimering af timingen og koncentrationen af polariserende reagenser. Optimering alle disse faktorer bør føre til informative resultater med dette system.
The authors have nothing to disclose.
This work was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) grant (BB/H018573/1) and a BD Biosciences grant.
RPMI | Sigma | R8758 | |
DMEM | Sigma | D5671 | |
Penicillin Streptomycin solution | Sigma | P4333 | |
L-Glutamine | Sigma | G7513 | |
β-mercaptoethanol | Sigma | M3148 | |
DPBS | Sigma | D8537 | |
MIG vector | Addgene | Plasmid 9094 | |
pCL-Eco vector | Addgene | Plasmid 12371 | |
Cell strainer | BD Falcon | 352350 | |
Magnetic beads mouse CD4 cell kit | Invitrogen (Dynabeads) | 11415D | |
Streptavidin Beads | Miltenyi Biotech | 130-048-102 | |
MS cell separation columns | Miltenyi Biotech | 130-042-201 | |
LS cell separation columns | Miltenyi Biotech | 130-042-401 | |
CD25 Biotenylated MAb | BD Biosciences | 85059 | clone 7D4 |
CD62L Biotenylated MAb | BD Biosciences | 553149 | clone MEL-14 |
Polybrene (Hexadimethrine Bromide) | Sigma | 107689 | |
Anti-CD3 | eBiosciences | 16-0031-85 | clone 145-2C11 |
Anti-CD28 | eBiosciences | 16-0281-85 | clone 37.51 |
Anti-IL-4 | BD Biosciences | 559062 | clone 11B11 |
Anti-IFN-gamma | BD Biosciences | 559065 | clone XMG1.2 |
Anti-IL-2 | BD Biosciences | 554425 | cloneJES6-5H4 |
Recombinant IL-12 p70 | eBiosciences | 14-8121 | |
Recombinant IL-4 | BD Biosciences | 550067 | |
Recombinant TGF-beta | eBiosciences | 14-8342-62 | |
Recombinant IL-6 | eBiosciences | 14-8061 | |
Recombinant IL-2 | eBiosciences | 14-8021 | |
PMA | Sigma | P8139 | |
Ionomycin | Sigma | I0634 | |
Brefeldin A | eBiosciences | 00-4506 | |
Paraformaldehyde | Sigma | 16005 | Paraformaldehyde is toxic so use appropriate caution when handling |
Foxp3 staining buffer set | eBiosciences | 00-5523 | |
Anti-CD4 FITC | eBiosciences | 11-0041 | clone GK1.5 |
Anti-CD8a perCP-cy5.5 | eBiosciences | 45-0081-80 | clone 53-6.7 |
Anti-MHCII PE | eBiosciences | 12-0920 | clone HIS19 |
Anti-CD25 PE | eBiosciences | 12-0251-82 | clone PC61.5 |
Anti-CD62L PE | eBiosciences | 12-0621-82 | clone MEL-14 |
Anti-CD44 APC | eBiosciences | 17-0441 | clone IM7 |
Anti-IFN-gamma FITC | eBiosciences | 11-7311-81 | clone XMG1.2 |
Anti-IL-4 PE | BD Biosciences | 554435 | clone 11B11 |
Anti-IL-9 PE or APC | eBiosciences/Biolegend | 50-8091-82/514104 | clone RM9A4 |
Anti-IL-17a PE | BD Biosciences | 559502 | clone TC11-18H10 |
Anti-Foxp3 APC or PE | eBiosciences | 17-5773-82/12-5773-80 | clone FJK-16s |
NaCl | Sigma | S7653 | |
KCl | Sigma | P9333 | |
Na2HPO4-2H2O | Sigma | 71643 | |
Dextrose/Glucose | Sigma | G7021 | |
HEPES, free acid | Sigma | H3375 | |
NH4Cl | Sigma | A9434 | |
Disodium EDTA | Sigma | D2900000 | |
KHCO3 | Sigma | 237205 | |
CaCl2 | Sigma | C5670 |