Genetic Modification of Primary Human Myeloid Cells to Study Cell Migration, Activation, and Organelle Dynamics

Abstract

Myeloid dendritic cells (DCs) and macrophages are mononuclear phagocytes with key roles in the immune system. As antigen-presenting cells, they link innate detection of microbes with programming adaptive immune responses. Myeloid DCs and macrophages also play critical roles in development, promote tissue homeostasis, and direct repair in response to injury and inflammation. As cellular migration and organelle dynamics are intimately connected with these processes, it is necessary to develop tools to track myeloid cell behavior and function. Here, we build on previously established protocols to isolate primary human myeloid cells from peripheral blood and report an optimized method for their genetic modification with lentiviral vectors to study processes related to cell migration, activation, and organelle dynamics. Specifically, we provide a protocol for delivering genetically encoded fluorescent markers into primary monocyte-derived DCs (MDDCs) and monocyte-derived macrophages (MDMs) to label mitochondria, peroxisomes, and whole cells. We describe the isolation of primary CD14+ monocytes from peripheral blood using positive selection with magnetic beads and, alternatively, isolation based on plastic adherence. Isolated CD14+ cells can be transduced with lentiviral vectors and subsequently cultured in the presence of cytokines to derive MDDCs or MDMs. This protocol is highly adaptable for cotransduction with vectors to knock down or overexpress genes of interest. These tools enable mechanistic studies of genetically modified myeloid cells through flow cytometry, fluorescence microscopy, and other downstream assays. © 2022 Wiley Periodicals LLC. Basic Protocol: Transduction of MDDCs and MDMs with lentiviral vectors encoding fluorescent markers Alternate Protocol 1: Isolation of monocytes by plastic adhesion Alternate Protocol 2: Transduction of MDDCs and MDMs with lentiviral vectors to knock down or overexpress genes of interest Support Protocol 1: Production and purification of lentiviral vectors for transduction into primary human myeloid cells Support Protocol 2: Flow cytometry of MDDCs and MDMs Support Protocol 3: Fixed and live-cell imaging of fluorescent markers in MDMs and MDDCs.

Keywords: gene delivery; innate immunity; viral vectors.

Figure 1.. Schematic illustration of monocyte isolation, genetic modification, and differentiation of MDDCs and MDMs.

The major steps of the protocols described in this article are shown (PBMC preparation, CD14+ cell isolation, transduction with lentiviral vectors, and differentiation into MDDCs or MDMs), corresponding with estimates of the time investment required at each step.

Figure 2.. Genetic modification of MDDCs with fluorescent lentiviral reporter vectors.

A) Representative flow cytometry histograms of mEmerald expression in MDDCs at day 7 after transduction with LKO-MCS (vector alone), mito-mEm, perox-mEm, or cell-mEm in the presence or absence of SIV-VLPs packaging Vpx. B) Geometric mean fluorescence intensity (gMFI) of mEmerald expression in transduced MDDCs. C) gMFI of CD86 expression in mock-treated or transduced MDDCs that were either unstimulated or stimulated with LPS (100 ng/ml for 24 h). For (C) and (D), n = 4 independent donors. D) Representative z-stack projections (merge of 10-20 planes, 0.5 micron step size) of live and fixed images of transduced MDDCs acquired with an LSM880 Airyscan Fast confocal microscope. Images reveal localization of fluorescent markers (mEmerald expression, green), nuclei (DAPI, blue), and actin (Phalloidin, magenta – only depicted in fixed cell images). Scale bar = 10 μm.

Figure 3.. Genetic modification of MDMs with fluorescent lentiviral reporter vectors.

A) Representative flow cytometry histograms of mEmerald expression in MDMs at day 7 after transduction with LKO-MCS (vector alone), mito-mEm, perox-mEm, or cell-mEm in the presence or absence of SIV-VLPs packaging Vpx. B) Geometric mean fluorescence intensity (gMFI) of mEmerald expression in transduced MDMs. C) gMFI of CD86 expression in mock-treated or transduced MDMs that were either unstimulated or stimulated with LPS (100 ng/ml for 24 h). For (C) and (D), n = 4 independent donors. D) Representative z-stack projections (merge of 10-20 planes, 0.5 micron step size) of live and fixed images of transduced MDMs acquired with an LSM880 Airyscan Fast confocal microscope. Images reveal localization of fluorescent markers (mEmerald expression, green), nuclei (DAPI, blue), and actin (Phalloidin, magenta – only depicted in fixed cell images). Scale bar = 10 μm.