Recording the wild lives of immune cells

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Science Immunology  07 Sep 2018:
Vol. 3, Issue 27, eaaq0491
DOI: 10.1126/sciimmunol.aaq0491


  • Fig. 1 Diagram of an intravital imaging setup.

    (Left) The equipment required for conducting an intravital imaging experiment includes an appropriate fluorescent mouse model, a microscope (shown here is an upright multiphoton microscope), laser sources, physiological monitoring, an appropriate anesthesia setup, and a tissue stabilizer to prevent motion artifacts during imaging. This image illustrates imaging in a dorsal skinfold window chamber; other tissues can be imaged (Fig. 2), each of them requiring their own methods of tissue preparation, stabilization, and monitoring. PMT, photomultiplier tube; APD, avalanche photodiode. (Right) Data acquisition, followed by data storage and data postprocessing to extract quantitative information.

  • Fig. 2 Existing applications of immune cell imaging in various tissues.

    (A) A wide range of mouse tissue sites is adapted for single-cell imaging. (B) Intravital micrograph showcasing detection of two immune cell types, namely, CD4+ T cells (red) and CX3CR1+ macrophages (green) in pancreatic islets. β cells (blue) were visualized with exendin-4 (Ex-4)–like neopeptide conjugated to the fluorescent dye Se-Tau-647, and the vasculature was detected with 500 K molecular weight dextran conjugated to a Pacific blue dye (gray). (C and D) Intravital micrographs showcasing detection of cytokines produced by immune cells in live mice, here in tumor tissues. (C) IFN-γ–producing lymphocytes (magenta). (D) IL-12b–producing myeloid cells (cyan). Tumor cells are also shown (gray).

  • Fig. 3 Single-cell imaging of drugs and their effects on immune cells.

    (A) Intravital imaging can reveal drugs’ (left) PK and (right) PD at single-cell resolution. The examples listed below indicate which types of drugs, cells, and responses can be detected and are relevant to immunology. (B) A PK study reveals that the immune checkpoint blocker anti–PD-1 mAb (aPD-1 mAb; gray) binds to T cells (green) only transiently in the tumor stroma (25). (C) A PD study reveals that radiation therapy primes tumor-associated macrophages (blue) to initiate vascular bursts. (With the permission of M. Miller, Massachusetts General Hospital, Boston.)



  • Table 1 Reporter systems to track different immune cells and their functions by single-cell imaging.

    This list serves as a general survey of intravital imaging labeling strategies across cell types; however, numerous Cre strains can additionally be incorporated into useable reporter systems. CFP, cyan fluorescent protein; DsRed, Discosoma sp. red fluorescent protein; FOXP3, forkhead box P3; TBX21, T-box 21; eGFP, enhanced GFP; Bcl6, B cell lymphoma 6; pDCs, plasmacytoid DCs; Zbtb46, zinc finger and BTB domain containing 46; Csf-1, colony-stimulating factor 1; AID, activation-induced cytidine deaminase; DTR, diphtheria toxin receptor.

    T cells
    GFP, CFP, DsRedRequires adoptive cell transfer(29)
    PA-GFPPhotoactivatable fluorescent reporter useful for cell tracking; requires adoptive
    cell transfer
    DPE -GFPA pan T cell marker; can also label pDCs(25)
    NFAT-GFP, NFAT-YFP;H2B-mCherryReadout of TCR signaling(43)
    IFN-γ–YFPSubset of activated T cells; can also be produced by NK cells(25)
    IL-10–GFPSubset of T cells; can also be expressed by macrophages(43)
    FOXP3-GFP, FOXP3-mRFPLabels Treg cells; FOXP3 is on the X chromosome, be aware of X chromosome
    inactivation in females
    Nur77-GFPLabels T cells and also B cells upon antigen receptor engagement(110)
    Granzyme B–TdTomatoA marker of granule exocytosis in cytolytic T cells and NK cells(111)
    Tbx21-ZsGreenFor assessing TH1 responses in T cells(112)
    CD2-eGFPLabels peripheral T cells and some NK subsets(113)
    IL-17F–CreeYFPMany variants exist for studying TH17 cells in vivo(114)
    IL-4–eGFPUsed to assess TH2 immunity(115)
    IL-21–GFPUsed to study the role of TFH cells(116)
    B cells
    GFP, CFP, DsRedRequires adoptive cell transfer(117)
    PA-GFPPhotoactivatable fluorescent reporter useful for cell tracking; requires adoptive
    cell transfer
    CD19-tdRFPExpressed by numerous B cells, adoptive transfer may be necessary if tissues are
    densely infiltrated by B cells
    Bcl6-YFPA B cell marker already validated for two-photon microscopy; also labels TFH cells(118)
    Blimp-1–YFPExpressed by B cells and highly expressed in plasma cells(119)
    Activation-induced cytidine deaminase (AID)–GFPSubset of B cells(120)
    CD11c(ITGAX)-YFP, CD11c-mCherryCells other than DCs can express CD11c(121)
    XCR1-Venus/+Homozygous mice are null for XCR1(55)
    XCR1-KIKGR/+Xcr1 replaced with photoconvertible fluorescent protein Kikume
    Green-Red (KikGR)
    CXCL9-RFP, CXCL10-BFPThese chemokines can also be expressed by other cell types, including monocytes,
    macrophages, granulocytes, and nonimmune cells
    SIGLEC-H–GFPExpressed by pDCs(54)
    Flt3-BFP2This construct has also been incorporated into a genetic model used to visualize
    DCs, macrophages, and CD4+ and CD8+ T cells simultaneously (Flt3-BFP2,
    Mertk-GFP–diphtheria toxin receptor, Cd4-tdTomato, Cd8a-tdTomato)
    Zbtb46-GFPA cDC marker that may be expressed in erythroid and endothelial populations(122)
    CX3CR1-GFP/+This chemokine receptor can also be expressed by T cells, NK, DC, and
    macrophages. Homozygous mice are null for CX3CR1
    CCR2-RFP/+Homozygous mice are null for CCR2(61)
    CX3CR1-GFP/+;CCR2-RFP/+Homozygous mice are null for CX3CR1 and/or CCR2(61)
    Mertk-GFP-DTRHomozygous mice are null for MerTK(11)
    c-fms–GFPc-fms is also known as Csf-1 receptor(60)
    MacBlueA gal4-responsive UAS-ECFP cassette also under the Csf-1 receptor promoter(124)
    Dye-conjugated ferumoxytolAdministered to mice several hours before imaging(25)
    Dye-conjugated dextrans and dextran nanoparticlesAdministered to mice several hours before imaging; can be used to label blood
    vessels in short term after administration
    Dye-conjugated anti-CD169 mAbUsed to label SCS macrophages(125)
    Dye-conjugated anti-F4/80 mAbA pan macrophage marker in mice(61)
    Dye-conjugated anti–Ly-6G mAbImaging is performed shortly after administration (~15 min)(126)
    Dye-conjugated anti–Gr-1 mAbImaging is performed shortly after administration (~15 min)(126)
    LysM-GFPCan also be expressed by macrophages(77)
    Dye-conjugated anti-neutrophil elastase mAbImaging is performed shortly after administration (~15 min)(85)
    CXCL12-mRFPCan also be expressed by stromal cells(80)
    SYTOX GreenUsed to visualize NETs (extracellular DNA labeling); also demarcates cell death(85)
    Innate lymphocytes
    GFP, CFP, DsRedRequires adoptive cell transfer(92)
    CXCR6-GFPCan be used to track ILCs and iNKT cells but is also expressed by some T cells(89)
    Megakaryocytes and platelets
    PF4-cre tdTomatoLabels megakaryocytes as well as platelets, which are defined based on their
    smaller size
    Dye-conjugated anti-CD49b mAbLabels all platelets(85)
    CD41-YFPki/+Labels a fraction of platelets(85)
    HSC-derived cells
    GFP, CFP, DsRedRequires adoptive cell transfer and is used to track HSCs and their progeny(84)
    Nonimmune components
    Dye-conjugated anti–Lyve-1 mAbLabels lymphatics(45)
    AngioSPARKLabels blood vessels(71)
    Second-harmonic generationApplicable to multiphoton (not confocal) microscopy; enables visualization of
    collagen fibers
    IL-7–eCFPExpressed by stromal cells in thymus and bone marrow(44)
    GCaMP6sSensor of calcium flux; relevant in the context of, for example, virus-infected cells(39)
    qDotsBright, highly tunable fluorophores surface-engineered to maximize circulation(127)
    IsolectinUsed to label endothelial wall of blood vessels(128)
    Hoechst33342 and CellTracker dyesIndiscriminately labels cell nuclei or cytosol; often used for adoptive cell transfer(41)

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