Dendritic cells are important cells of the immune system and play a central role in HIV-1 infection as mentioned in the previous chapter. A good model to study dendritic cells are monocytes isolated from donor blood and differentiated into dendritic cells ex vivo by the addition of the cytokines granulocyte/macrophage colony stimulating factor (GM-SCF) and IL4. The so-called monocyte derived dendritic cells (MDDCs) have been shown to be a good model to study conventional DCs [271, 272]. The monocytes can also be differentiated into monocyte derived macrophages (MDMs) by adding only GM-SCF during differentiation. Once the cells are differentiated they are short lived and can be kept in culture only up to 5 to 10 days. Therefore conventional approaches for genetic manipulations (knockdown and ectopic expression of genes) have proven to be of limited success. For one they are resistant to transduction by lentiviral vectors (LVs) based on HIV-1 or MLV [273, 274] and difficult to transfect with siRNA. Not surprisingly siRNA transfection leads to the activation of the dendritic cells (see Figure 2.5), since they are specialized to detect PAMPs and siRNA resembles a putative RIG-I ligand. Once the discovery was made by Goujon et al. that Vpx not only increases infection of SIV but also HIV-1 in MDDCs and MDMs, the idea to use Vpx to transduce MDDC with HIV-1 based LVs was born [270, 275-277]. In 2010 the first paper was published by Manel et al. showing that newly produced capsid is sensed in a CypA dependent manner when more than 80%
of an MDDC culture is infected with HIV-1 with the help of SIVMAC virus like particles containing Vpx (Vpx-VLPs). At the same time an IRF3 knockdown was achieved in MDDCs using Vpx-VLPs to transduce the cells with a LV knockdown construct [147].
We adapted the method to achieve MDDC transduction with our own knockdown vectors (see chapter 3-5 for detailed methods). Freshly isolated CD14+ monocytes from healthy donor buffy coats (leukocyte fraction of blood donation) were isolated using magnetic beads couple to anti-CD14 antibodies and incubated with Vpx-VLPs for 2 to 3 hours (Figure 2.1 A). Then the monocytes were transduced with HIV-1 based LV expressing a micro RNA 30 (miR30) based short hairpin RNA (shRNA) either targeting the gene of interest or targeting the firefly luciferase gene (Luc) as control. The miR30 cassette is followed by a puromycin selection marker or a GFP sequence to monitor LV expression levels after transduction by FACS analysis (Figure 2.1 D).
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The miR30 back bone and the selection cassette are transcribed in the same transcript by RNA Polymerase II (Pol II) and the shRNA is matured by the cellular micro RNA maturation system [278]. LVs pseudotyped with the envelope protein of vesicular stomatitis virus (VSVg) and Vpx-VLPs preincubation proved to be the most effective way to transduce monocytes. LVs pseudotyped with the feline endogenous retrovirus envelope protein RD114 was not able to transduce monocytes even in the presence of Vpx-VLPs (Figure 2.1 B). It is possible to incorporate Vpx into HIV-1 particles either unspecifically or by modifying the HIV-1 p6 sequence to bind and incorporate Vpx specifically [139, 279]. Providing Vpx in trans to LVs increased the efficiency of MDDC transduction rate (Figure 2.1 C) but was not superior to pre-treatment with Vpx-VLPs. We also used a LV packaging plasmid with a modified p6 incorporated Vpx but it did not result in the desired particle production yield (data not shown). Treating monocytes with Vpx-VLPs yielded up to 90% of transduced MDDCs after 4 days of differentiation (Figure 2.1 D). The best transduction efficiency was achieved using a 1:3 ratio of Vpx-VLP supernatant combined with a 1:4 of LV supernatant (both >1 mU RT/µl, corresponding to an approximate titer of >106/ml on HEK 293 cells) with a final cell concentration of 2x106/ml [147, 280, 281]. HIV-1 is able to infect monocytes but the expression of the provirus is delayed and is only expressed 2 to 3 days after the differentiation is induced (Figure 2.1 D). The lack of provirus transcription has been associated to the absence of the host cyclin T1 protein (CycT1) recruited by Tat to initiated transcription. CycT1 appears to be controlled by miR198 ([282] and reviewed here [283]). Since GFP expression of the LV construct used is not under the control of the HIV-1 LTR, there must be additional blocks to HIV-1 provirus transcription in monocyte and they disappear during dendritic cell differentiation. Additionally, HIV-1 reverse transcription in monocytes seems to be slower even in the presence of Vpx-VLPs since PCR for LRT products and 2-LTR circles could only be measured 3 days after infection of monocytes with a HIV-1 3 part reporter virus (see chapter 5).
Monocytes are characterized by expressing CD14 (together with MD2 the co-receptor of TLR4) and have the capacity to differentiated into macrophages or tissue specific dendritic cells depending on the cytokine stimulation. The differentiation from monocytes to MDDCs is characterized by upregulation of DC-SIGN and CD1a and
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downregulation of CD14. Also the activation maker CD86 expressed on monocytes is downregulated during MDDC differentiation.
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CD86 also known as B7-2 is upregulated once immature dendritic cells have detected a PAMP and serves together with CD83 as a marker for mature dendritic cells. CD86 (together with CD80/B7-1) is the receptor for CD28 on CD4+ T cells and important to activate T cells during antigen presentation. The role for both CD1a and CD83 is poorly understood. Increasing amounts of Vpx-VLPs and LV resulted in more GFP positive cells but no activation was observed, monitored by CD86 and CD83 FACS analysis (Figure 2.2 A middle panel and 2.4).
The expression of the MDDC marker DC-SIGN was normal (Figure 2.4) with more than 90% of DC-SIGN positive MDDC after 4 days of differentiation. However, CD1a surface staining revealed that with increasing amounts of Vpx-VLPs and LVs the fraction of CD1a positive MDDC was decreasing (Figure 2.2 A bottom panel). Normal differentiation resulted in >60% of CD1a positive MDDC but less than 20% of MDDC stained for CD1a surface expression after differentiation in presence of Vpx-VLPs and LVs. Activation of immature MDDC after transduction with lentiviral vector at very high MOI has been reported as well [284]. Interestingly, the phenotype resembles MDDCs differentiated in presence of IFNβ. When monocytes were stimulated with 0.1 ng/ml to 10 ng/ml of recombinant IFNβ the MDDCs showed upregulation of CD86 and downregulation of CD1a in a dose dependent manner (Figure 2.2 B). The low CD1a expression was accompanied with a lack of downregulation of CD14 expression (data not shown) indicating that activated monocytes were not properly differentiated into MDDCs but retained monocyte/macrophage characteristics (CD1a-, CD14+ and CD86+).
The knowledge that Vpx is degrading SAMHD1 in myeloid dendritic cells leading to the increase of the intracellular dNTP pool promted us to adapt the above described protocol. Instead of treating the monocytes with Vpx-VLPs they were incubated with a mix of all four nucleosides (dA, dG, dC and T) for 2 hours and then transduced with a GPF encoding LV (Figure 2.3 A). Incubating the monocytes with 2.5 mM nucleoside mix resulted in 60% to 70% transduced MDDC after 4 days (Figure 2.3 B). Transduction of monocytes using nucleosides did not reached the levels achieved by Vpx-VLPs assisted transduction and showed slower kinetics in GPF expression after transduction since Vpx-VLPs allow higher levels of provirus integration (see chapter 5). Transduction levels with nucleosides were not high enough to achieve expression levels of the LV construct for gene knockdown but
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were sufficient for ectopic expression of genes of interest (Figure 2.3 C and chapter 5). 5 days after transduction with a vector expressing a red fluorescent Myc-tagged dsRED protein (see Annex I Table 5 for Myc-dsREDexpress sequence), 7.44% of the MDDCs showed expression (Figure 2.3 C) and the myc-tagged protein could be detected by western blot analysis (Figure 2.3 D). The percent of transduced cells is probably underestimated since the red fluorescent signal of dsRED is weaker compared to GFP. Similar experiments with GFP expression yielded up to 50% to 70% positive cells (compare to Figure 2.3 B and data not shown). The red protein has been chosen to allow simultaneously expression of dsRED as a control protein for ectopic gene expression and infection with an HIV-1 reporter vector expressing GFP.
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Comparisons of monocytes treated either with Vpx-VLPs or nucleoside revealed similar numbers of DC-SIGN positive MDDC, although cells treated with nucleoside were below 90% and the peak of fluorescent was shifted to the left indicating lower DC-SIGN expression (Figure 2.4). The same effect was also observed for CD1a expression were the nucleoside treatment resulted in less CD1a positive MDDC after 5 days of differentiation. This is probable due to the toxic effect of nucleosides. 2.5 mM to 3 mM is highest concentration tolerated by monocytes. The use of 5 mM nucleoside concentration led to considerable increase in cell death judged by a propidium iodide stain (data not shown). Interestingly, both Vpx-VLPs and nucleosides transduced MDDCs showed lower upregulation of CD83 compared with untreated MDDC after stimulation with 100 ng/ml of LPS to induce maturation (Figure 2.4).
The use of siRNA transfection has been reported to work to knockdown genes in MDDC by Nikolic et al. [285, 286]. We used the same protocol which consists of two rounds of transfection of 20 nM siRNA in immature MDDC to achieve knockdown of SAMHD1 (see chapter 5). Knockdown of the protein was successful but at the same time siRNA induced the maturation of the MDDCs. CD83 expression was induced after siRNA transfection indicating MDDC activation (Figure 2.5 A). Indeed MX1 expression was greatly increased in MDDC transfected with siRNA or just treated with the transfection lipid alone (HiPerfect). MX1 levels were even higher than in untreated MDDCs stimulated with 100 ng/ml LPS (Figure 2.5 B). We tested several transfecting reagents (RNAiMax and Lipofectamin from Invitrogen and INTERFERin from Polyplus) and all of them were activating MDDCs (data not shown). For experiments where the maturation state of the MDDCs is not relevant or all cells are matured by LPS for example siRNA can be used to achieve gene knockdown but in experiment where immature cells are desired or immature cells are compared to mature cells, siRNA is not the method of choice.
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