Here we provide a protocol for screening potential transcription factors involved in the development of dendritic cell (DC) using lentiviral transduction of shRNA to obtain stable knockdown cell lines for in vitro DC differentiation.
Dendritic cells (DCs) are important antigen-presenting cells that connect innate and adaptive immune responses. DCs are heterogeneous and can be divided into conventional DCs (cDCs) and plasmacytoid DCs (pDCs). cDCs specializes in presenting antigens to and activate naïve T cells. On the other hand, pDCs can produce large quantities of type I interferons (IFN-I) during viral infection. The specification of DCs occurs at an early stage of DC progenitors in the bone marrow (BM) and is defined by a network of transcription factors (TFs). For example, cDCs highly express ID2, while pDCs highly express E2-2. Since more and more subsets of DCs are being identified, there is a growing interest in understanding specific TFs controlling DC development. Here, we establish a method to screen TFs critical for DCs differentiation in vitro by delivering lentivirus carrying short hairpin RNA (shRNA) into an immortalized hematopoietic stem and progenitor cell (iHSPCs) line. After the selection and in vitro differentiation, cDC and pDC potential of the stable knockdown cell lines are analyzed by flow cytometry. This approach provides a platform to identify genes potentially governing DC fates from progenitors in vitro.
DCs are key regulators of innate and adaptive immunity1. DCs are mainly classified into two functionally distinct populations, namely pDCs and cDCs. Moreover, cDCs comprise two subsets, namely, type I and type II cDCs or cDC1s and cDC2s, respectively2. pDCs, expressing BST2, Siglec-H, and intermediate levels of CD11c in mice3,4, are the cells that can secrete large amounts of IFN-I during inflammation and viral infection5. Due to their robust IFN-I-producing ability, they are also suspected to play a key role in the progression of autoimmune diseases, including systemic lupus erythematosus (SLE)6. cDC1s, defined by the surface expression of XCR1, CD8a, CLEC9A, and CD103 in mice7, are specialized in the activation and polarization of cytotoxic CD8+ T cells (CTLs) through the antigen cross-presentation, thereby initiating type I immunity in response to intracellular pathogens and cancer8,9. On the other hand, cDC2s, expressing CD11b and CD172α (also known as Sirpα) in both humans and mice, can activate CD4+ T cells and promote type II immune response against allergen and parasites10, as well as modulate type III immunity following extracellular bacteria and microbiota recognition11,12.
Diversification of DCs is determined by a group of TFs from hematopoietic stem and progenitor cells (HSPCs) in the BM. E2-2 (encoded by Tcf4) is a master regulator for differentiation and function of pDCs13,14. In contrast, the inhibitor of DNA binding 2 (ID2) drives cDC specification and inhibits pDC development through blocking E protein activity15. Moreover, the development of cDC1s requires IRF8 and BATF3, while differentiation of cDC2s highly depends on IRF416. Recent works have explored the heterogeneity of pDCs17 and cDCs and their transcriptional regulation18. Because of the complexity of DC network, there is an unmet need to establish a platform to identify other TFs controlling the development and functionality of DCs.
Here, we used an iHSPC that was generated by expressing estrogen-regulated nuclear translocation of Hoxb8 in BM cells (also referred to as Hoxb8-FL cells)19. iHSPCs can proliferate and remain in an undifferentiated stage in the presence of β-estradiol and Flt3 ligand (FL), whereas they start to differentiate into different DC types in the presence of FL upon withdrawal of β-estradiol19. Based on this feature, we can knock down genes of interest at the progenitor stage, followed by examining the effect on in vitro differentiation of pDCs and cDCs. Therefore, this method is a powerful tool to discover the genes that regulate the development and function of DCs.
Handling of lentivirus is performed as per the regulation of the Department of Environmental Health and Safety of National Taiwan University College of Medicine.
1. Preparation of immortalized hematopoietic stem and progenitor cell lines (iHSPCs)
2. Lentiviral transduction
3. Measurement of knockdown efficiency by reverse transcription and real-time PCR (RT-PCR)
4. In vitro differentiation of the stable knockdown iHSPC cell lines
5. Flow cytometric analysis of the differentiated DCs
The map of lentiviral vector pLKO.1-Puro is shown (Figure 1). After the delivery of lentivirus expressing shRNA against LacZ (a non-targeting control), Tcf4, and Id2 in iHSPCs, the knockdown efficiency confirmed by RT-qPCR revealed that the expression of Tcf4 was reduced in shTcf4 iHSPCs, compared to shLacZ iHSPCs (Figure 2A). On the other hand, the decreased expression of Id2 was also observed in shId2 iHSPCs, compared to shLacZ iHSPCs control (Figure 2B). The shLacZ,shTcf4, and shId2 iHSPCs cell lines were differentiated into pDCs and cDCs in vitro with FL. After five-day culture of shLacZ iHSPCs, the frequency of CD11c+ cells, which represent DC population7, was around 95% (Figure 3A, left panel). However, knockdown of Tcf4 or Id2 slightly decreased the generation of CD11c+ DCs (Figure 3A, middle and right panel). Moreover, further analysis of CD11c+ DCs revealed that shLacZ iHSPCs differentiated into 70% of cDCs (CD11c+CD11b+B220+) and 22% of pDCs (CD11c+CD11b–B220+) (Figure 3B, left panel). However, shTcf4 iHSPCs generated a significantly lower percentage of pDCs (4 %) than did shLacZ control (Figure 3B, middle panel). On the other hand, shId2 iHSPCs generated a significantly lower percentage of cDCs (54%) but a higher percentage of pDCs (39%) than did shLacZ control (Figure 3B, right panel). Therefore, these results suggest iHSPCs faithfully reflect the same requirement of transcription factors for controlling DC development.
Figure 1: The construct of lentiviral vector pLKO.1-Puro. Please click here to view a larger version of this figure.
Figure 2: The knockdown efficiency of shTcf4 and shId2. After stable gene knockdown, RNAs were isolated from shLacZ, shTcf4, and shId2 iHSPCs and reversely transcribed into cDNA, and the expression of Tcf4 and Id2 was measured by RT-qPCR. Relative gene expression was normalized to Rpl7. A. The expression of Tcf4 in shLacZ and shTcf4 iHSPCs. B. The expression of Id2 in shLacZ and shId2 iHSPCs. Please click here to view a larger version of this figure.
Figure 3: Knockdown of Tcf4 impairs pDC development, whereas knockdown of Id2 reduces cDC generation from iHSPCs in vitro. iHSPCs were stably transduced with shLacZ, shTcf4, and shId2-carrying lentiviruses, then in vitro differentiated into DCs with FL (100 ng/mL). After culture for 5 days, the cells were stained and analyzed by flow cytometry. A. The percentage of CD11c+ cells is shown. B. The analysis for pDCs (CD11c+B220+CD11b–), and cDCs (CD11c+B220–CD11b+) are shown. Please click here to view a larger version of this figure.
PCR reaction | |||
Steps | Temperature | Time | Cycles |
Initial Denaturation | 95°C | 3 minutes | 1 |
Denaturation | 95°C | 15 seconds | 35-40 |
Annealing | 60°C | 20 seconds | |
Extension | 72°C | 20 seconds | |
Final Extension | 80°C | 20 seconds | 1 |
Hold | 4°C | ∞ | 1 |
Table 1: Thermocycler conditions for PCR.
Lentivirus-based shRNA vectors are often used for gene silencing by viral transduction into cells and permit stable integration into the host genome. However, various transduction efficiency in different cell types needs to be considered, and a number of approaches have been taken to overcome this problem.
Polybrene is a polycationic polymer that can neutralize the charges on the cell membrane, thereby enhancing the binding of the virion to the cells during transduction20. Although it is an effective way to increase the transduction rate, it is also toxic to some cell types when adding excessive amounts. In this case, it is required to test the toxicity of polybrene and optimize the concentration in different cells. Protamine sulfate, a cationic compound, maybe an alternative approach to increase cell viability21. Moreover, refresh complete media on the same day of infection can improve the represent survival rate after transduction.
Most shRNA delivery systems have a selective marker for eliminating cells that have not been successfully infected. The lentiviral system we used contains a puromycin-resistant gene so that the infected cells could be selected after transduction. Moreover, lentivirus expressing GFP is also another option for the selection. However, there are both advantages and disadvantages to both methods. Using puromycin as a selection method is hard to directly measure the transduction efficiency unless the expression of target genes was confirmed by qPCR. But puromycin provides stress for selection to keep the foreign DNA inside the cells and maintain the knockdown phenotype. In contrast, GFP is also a selection method that does not provide stress to the cells even though the transduction efficiency can be immediately evaluated by flow cytometry.
One of the limitations of this method is the relatively small number of cDC1 cells generated by iHSPCs in vitro19. Although iHSPCs have pDCs and cDCs potential driven by FL in vitro, most of cDC subtypes generated is cDC2s, but not cDC1s. This is likely due to the requirement of Notch signaling during in vitro cDC1 differentiation22. Therefore, co-culture with OP9-DL1 stromal cells which express the Notch ligand Delta-like 1 may restore cDC1 potential of iHSPCs, thereby improving the mechanistic studies on cDC1s.
The application of this protocol not only for investigating the role of TFs but also for other genes, like metabolic genes likely to participate in the development of DCs or other cell types. An emerging concept highlights that DC developmental pathway is associated with different cellular metabolism23,24. Therefore, gene knockdown in DC progenitors is a powerful strategy to study metabolic regulation in response to environmental cues and determine how different metabolic pathways regulate DC differentiation by knocking down critical genes in metabolism25. On the other hand, iHSPCs have the capacity to differentiate into myeloid cells beyond DC lineages by utilizing specific differentiation factors. The use of macrophage colony-stimulating factor (M-CSF) and granulocyte-colony stimulating factor (G-CSF) results in the development of macrophages and granulocytes, respectively, from iHSPCs19. Based on the same strategy, this method could also apply to the research of myeloid cell development.
Collectively, the described protocol from gene knockdown to in vitro differentiation of iHSPCs provides a rapid and effective way to facilitate the study of DC development and answers fundamental questions of immune cell development in the future.
The authors have nothing to disclose.
We are grateful for technical support from Dr. Tz-Ling Chen. We thank the National RNAi Core Facility (Academia Sinica, Taiwan) for providing shRNA lentivirus (http://rnai.genmed.sinica.edu.tw). This work was supported by the Ministry of Science and Technology, Taiwan (MOST 108-2320-B-002-037-MY3 and MOST 109-2320-B-002-054-MY3).
Antibodies | |||
APC/Cy7 anti-mouse CD11c Antibody | Biolegend | 117324 | (Clone: N418) |
FITC anti-mouse/human CD11b Antibody | Biolegend | 101206 | (Clone: M1/70) |
PE anti-mouse/human B220 Antibody | Biolegend | 103208 | (Clone: RA3-6B2) |
Cell culture | |||
1.5 mL Micro tube | ExtraGene | TUBE-170-C | |
12-well tissue culture-treated plate | Falcon | 353043 | |
Fetal bovine serum (FBS) | Corning | 35-010-CV | |
RPMI 1640 medium | gibco | 11875-085 | |
Reagent | |||
β-estradiol | Sigma-Aldrich | E2758-250MG | |
β-mercaptoethanol (β-ME) | Sigma-Aldrich | M6250 | |
FACS buffer | home-made | Formula: 1xPBS+0.5 %FBS+0.1%NaN3 | |
Flt3 ligand (FL) | home-made | ||
Polybrene | Sigma-Aldrich | TR-1003-G | |
Puromycin | Invivogen | ant-pr-1 | |
TRIsure | BIOLINE | BIO-38032 | |
shRNA (Taregt sequence/clone ID) | Company | ||
shId2 (GCTTATGTCGAATGATAGCAA/TRCN0000054390) | The RNAi Consortium (TRC) | ||
shLacZ (CGCGATCGTAATCACCCGAGT/TRCN0000072224) | The RNAi Consortium (TRC) | ||
shTcf4 (GCTGAGTGATTTACTGGATTT/TRCN0000012094) | The RNAi Consortium (TRC) |