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.

CSPG4-targeting CAR-macrophages inhibit melanoma growth

“…1C). Using our previously established protocols (33), CSPG4-CAR expressing macrophages were generated with high transduction efficiencies (Supplemental Fig. 2B), and thus we proceeded with using primary macrophage-derived CAR-Ms for all subsequent studies.…”

“…pCMV-VSV-G, psPAX2, and transgene plasmids were transfected into HEK293FT cells to generate lentivirus media as previously described (33) and concentrated using Lenti-X concentrator (Takara Bio #631232) according to the manufacturers protocol.…”

“…Anonymous donor healthy human blood in either leukoreduction filters or apheresis cones were obtained from Associated Regional and University Pathologists, Inc (ARUP). Leukocytes were recovered from blood, CD14+ monocyte isolated by adhesion, transduced, and cultured as previously described (33).…”

Transferred mitochondria accumulate reactive oxygen species, promoting proliferation

“…PBMCs were isolated from leukocyte filters obtained from de-identified human blood donors (ARUP Blood Services). CD14 +monocytes were isolated from buffy coats and genetically modified with lentiviral vectors in the presence of virus-like particles packaging Vpx (to overcome restriction in myeloid cells) as previously described ( Johnson et al, 2020 ; Greiner et al, 2022 ). Briefly, freshly harvested CD14 +monocytes were plated at a density of 4–5 M cells per 10 cm plate in ‘macrophage culture media’ containing: RPMI (11875119, ThermoFisher), 10% FBS (26140079, Thermo Fisher), 0.5% penicillin/streptomycin (P/S; P4333, Thermo Fisher), 10 mM HEPES (15630080, ThermoFisher), 0.1% 2-Mercaptoethanol (21985023, Thermo Fisher), recombinant human GM-CSF at 20 ng/ml (300–03, Peprotech) with the addition of polybrene (1 μg/ml), and supernatant containing Vpx particles (0.5 mL per 4 M cells) to facilitate viral transduction.…”

Cell-free assays reveal that the HIV-1 capsid protects reverse transcripts from cGAS

“…To generate MDDCs and MDMs, we acquired leukocytes from de-identified normal human donors from ARUP Blood Services, Sandy, UT, USA, similar to previously described methods 62 . We cannot report on the sex, gender, or age of the donors since the samples were de-identified and donors remain anonymous.…”