Abstract
Our protocol describes adoptive transfer of antigen presenting cells (APCs) isolated from the lungs by enzymatic digestion and magnetic enrichment. This protocol can be used to study APC functions and trafficking.
Keywords: Lung antigen presenting cells, Dendritic cells, Macrophages, Adoptive transfer, Enzymatic digestion, Magnetic cell isolation, Intravenous injection
Background
Lung APCs, including macrophages and dendritic cells (DCs), play a critical role in sensing invading pathogens, priming T cell responses and controlling tolerogenic responses. Lung DCs are the most potent professional APCs, including conventional DCs (cDCs) and plasmacytoid DCs (pDCs) during steady-state, and newly recruited monocyte-derived DCs (moDCs) upon inflammation (Kopf et al., 2015). Lung resident macrophages, including alveolar macrophages, interstitial macrophages and bronchial macrophages, are less potent in presenting antigens. All macrophages and cDCs express high levels of CD11c on their cell surface, while pDCs express intermediate levels of CD11c (Becher et al., 2014). Thus, CD11c magnetic microbeads can be used to isolate mouse lung macrophages and cDCs. We developed a protocol of isolating lung APCs from normal mice and then adoptively transferring them to syngeneic bone marrow transplanted mice. We used this protocol to determine whether normal lung APCs are sufficient to restore T helper cell polarization in response to herpesvirus infection post-transplant (Zhou et al., 2016).
Materials and Reagents
Equipment
Procedure
Data analysis
Single cell suspension was prepared from the lungs of C57BL/6 mice three days post infection of murine gamma herpesvirus 68 (MHV-68) intranasal at a dose of 5 x 104 pfu/mouse (Zhou et al., 2016). CD11c+ cells were then enriched by using anti-mouse CD11c microbeads. To analyze the composition of these microbead-enriched cells by flow cytometry, appropriate fluorescence-conjugated antibodies were used to stain cell surface markers. Percentages on each plot represent the percentages of each parental gate. Similar results were obtained in two additional independent experiments. The majority enriched CD11c+ APCs are alveolar macrophages (AMs) (CD45+ CD11c+ MHC IIint CD64hi), and DCs. The DC population can be further separated into CD103 cDCs (CD45+ CD11c+ MHC IIhi CD64- CD103hi CD11bint), CD11b+ cDCs (CD45+ CD11c+ MHC IIhi CD64- CD103lo CD11bhi) and MoDCs (CD45+ CD11c+ MHC IIhi CD64+ CD103lo CD11bhi) (Misharin et al., 2013; Wang et al., 2006) (Figure 3) . Lung APCs were prepared from congenic CD45.1 mice three days post infection of MHV-68, and adoptively transferred into wild-type C57BL/6 mice (CD45.2+, but CD45.1-). The recipients were infected with MHV-68 24 h after cell transfer, and euthanized for flow cytometry analysis 48 h after cell transfer. Representative flow cytometry plots show donor CD45.1+ APCs in the lungs, lung draining lymph nodes (dLN) and spleens of CD45.1- recipients (Figure 4). Figure 3. Representative flow cytometry plots of APCs isolated from mouse lungs Figure 4. Tracking injected cells. Upper panel, wild-type C57BL/6 mice without cell transfer; lower panel, wild-type C57BL/6 recipients adoptively transferred with CD45.1+ lung APCs.
Notes
If CD11c+ subpopulations are under investigation, the magnetically enriched cells can be further stained with appropriate fluorescence-conjugated antibodies and sorted to specific APC populations using fluorescence-activated cell sorting (FACS) (Misharin et al., 2013).
Recipes
Acknowledgments
This protocol was adapted from our publication (Zhou et al., 2016). This work was supported by NIH grants AI117229, HL115618, T32HL07749 and 2UL1TR000433.
References
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