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Caught in the Act: Intravital Multiphoton Microscopy of Host-Pathogen Interactions

2009; Cell Press; Volume: 5; Issue: 1 Linguagem: Inglês

10.1016/j.chom.2008.12.007

1934-6069

Heather D. Hickman, Jack R. Bennink, Jonathan W. Yewdell,

Advanced Biosensing Techniques and Applications

Intravital multiphoton microscopy provides a unique opportunity to discover and characterize biological phenomena in the natural context of living organisms. Here we provide an overview of multiphoton microscopy with particular attention to its application for studying host-pathogen interactions. Intravital multiphoton microscopy provides a unique opportunity to discover and characterize biological phenomena in the natural context of living organisms. Here we provide an overview of multiphoton microscopy with particular attention to its application for studying host-pathogen interactions. Humans, like all jawed vertebrates, are confronted with hordes of pathogens seeking a host in which to procreate. Thanks to evolution, we are endowed with a highly sophisticated multilayered response that utilizes numerous strategies for preventing, limiting, and eradicating infections. The initial response is mounted by the innate immune system, which plays a crucial holding action for up to a week to limit pathogen replication to manageable levels. This involves soluble (e.g., interferons) and cellular (e.g., natural killer [NK] cells) elements of the innate immune system. The adaptive immune system initiates lymphocyte responses to generate effector T cells and antibodies within a few hours of infection. When successful, the immune system eliminates the threat and the host survives to pass on its own genes to future generations. Although there has been tremendous progress in understanding immunity to pathogens, gained in large part through ex vivo approaches, many questions remain. When and where is infection established at the cellular level? How does infection disseminate through the organ/organism? How do infected cells signal to the immune system, and how quickly after infection do cells of the innate immune system respond? How do innate responses to infection shape adaptive responses? How and where do immune effector cells encounter pathogens and/or pathogen-infected cells? What happens next? How do the answers differ between pathogens? A long list to be sure, but longer still as each answer raises its own set of questions. The most direct approach to understanding the complex cellular events occurring at the organismal level after infection is to simply look at an infection as it progresses. Previously, direct visualization of infection has been essentially limited to static immunofluorescence confocal microscopy imaging of sectioned tissues. Lately, the advent of new technologies such as two-photon (2P) microscopy and whole-body imaging have provided new perspectives on both pathogen behavior and host responses within the live host. In this Review, we provide a broad overview of 2P microscopy as it relates to imaging infectious organisms, focusing on the benefits, caveats, and pitfalls of this methodology. From analysis of virion release from cells on a coverslip to imaging bacterial invasion of an entire organism, light microscopy has rapidly advanced our understanding of host-pathogen interactions. Until recently, fluorescence microscopy relied on single-photon excitation—i.e., a photon of a given wavelength excites a fluorophore, resulting in emission of a longer wavelength photon that is then registered by a detector, be it the human retina, film, or photomultiplier array (Figure 1A). In wide field epifluorescence microscopy, the entire microscope field is bathed in fluorescent light, and fluorescent molecules in the optical path are equally excited and detected regardless of their relationship to the focal plane. The distinguishing feature of confocal microscopy is the addition of a confocal pinhole that greatly reduces out-of-focus fluorescence. The result is greatly enhanced image quality, and the ability to computationally generate 3D images by collecting images as the focal plane is precisely moved in the z direction by raising or lowering the microscope objective. Laser scanning confocal microscopy (LSCM) has provided a solid foundation for our understanding of cellular events occurring after infection. Sections can be cut from infected tissues and analyzed for the presence of pathogens as well as immune cell subsets (even endogenous antigen-specific lymphocytes [Khanna et al., 2007Khanna K.M. McNamara J.T. Lefrancois L. In situ imaging of the endogenous CD8 T cell response to infection.Science. 2007; 318: 116-120Crossref PubMed Scopus (96) Google Scholar]). LSCM has numerous advantages over other ex vivo techniques: (1) instruments, while expensive, are widely available, typically at core facilities that provide expertise and services at affordable rates; (2) it provides an actual image of cell interactions occurring postinfection; (3) a veritable rainbow of colors can be used for imaging, allowing multiple antibodies and stains to be used, including those requiring cell permeabilization, and (4) imaging can be performed at the convenience of the investigator since sections can typically be stored indefinitely. The adaptability to a wide variety of experimental situations has made confocal microscopy the method of choice for many different studies of host-pathogen interplay. Along with its numerous advantages, however, come some drawbacks: (1) LSCM provides only a static image, making it difficult (at best) and frequently impossible to identify transient events and impossible to observe the movement of immune cells; (2) tissue sectioning can have profound effects on tissue morphology and antigenicity; (3) generating overlapping sections to cover large areas of tissues is technically challenging and labor intensive, as is generating serial sections to overcome the limited working distance of the focal plane (about 100 μM can be imaged in the z plane). Enter two-photon fluorescence laser scanning microscopy (2PLSM) (Denk et al., 1990Denk W. Strickler J.H. Webb W.W. Two-photon laser scanning fluorescence microscopy.Science. 1990; 248: 73-76Crossref PubMed Scopus (8288) Google Scholar), which uses a high-power laser to deliver short (∼100 femtosecond), rapid (∼80 MHz), and intense (∼109 Watts/mm2) pulses of lower-energy (near infrared) photons to a tissue of interest. When two photons hit a fluorophore within an attosecond (10−18) of each other, they combine their energies to excite the fluorophore to a state achieved by a single photon with approximately twice the energy (Figure 1B) (Zipfel et al., 2003Zipfel W.R. Williams R.M. Webb W.W. Nonlinear magic: multiphoton microscopy in the biosciences.Nat. Biotechnol. 2003; 21: 1369-1377Crossref PubMed Scopus (3152) Google Scholar). High power is needed, since 2P excitation requires a million-fold higher photon flux than standard 1P excitation to activate the same number of fluors (3P excitation is possible since only a 10-fold higher photon flux is required). 2P excitation mode confers significant advantages over standard LSCM. Longer wavelengths scatter less and are not absorbed as readily by endogenous chromophores, resulting in deeper tissue penetration (up to 1 mm in some studies) and less phototoxicity than standard 1P excitation. Since simultaneous absorption of two photons is highly dependent on photon flux, and essentially only occurs at the focal plane of the objective, confocality is achieved without using a pinhole, increasing detection sensitivity and limiting photoxicity to the focal plane itself (Figure 2). These factors make 2P microscopy the choice for imaging living tissues. When combined with a new arsenal of fluorescent pathogens, this technique enables imaging of infections in real time in living animals. Leeuwenhoek, who first reported the microscopic world of bacteria and protists in 1676, would surely be impressed. There are myriad choices for 2P microscopes, ranging from turnkey off-the-shelf models to home-built custom instruments, with major microscope and laser manufacturers eager to contribute to (and profit from) 2P evolution. Understanding the underlying principles of MP and their application to living specimens will help you to select the microscope system and lasers best suited to your studies. As discussed above, 2PLSM relies on enormous photon fluxes, typically generated by Ti:sapphire lasers emitting ultrashort pulses of light (this is termed "mode locking"). Photon flux, which governs effective excitation (and imaging) depth, is expressed in terms of laser power. Laser power should be considered over the entire working range (in terms of wavelength) of the laser, as power typically peaks at 800 nm and declines precipitously with increasing and decreasing wavelengths. Although usable power in vital imaging is ultimately limited by phototoxicity, high power is needed for imaging at longer wavelengths (typically used to excite red fluorescent proteins [FPs]) and in applications such as photobleaching or uncaging, which demand intense excitation. Most Ti:sapphire lasers are tunable between ∼700 to 1000 nm but have the highest power output at 800 nm. While many fluorescent dyes can be excited at ∼800 nm, some red FPs require excitation at longer wavelengths of >1000 nm. On the horizon are ytterbium-doped lasers that should provide greater power at long wavelengths. Generating a sufficiently strong signal for deep-tissue imaging is a significant hurdle in 2PLSM. 2P lasers can deliver pulses ranging from picoseconds to femtoseconds, with shorter pulses carrying higher peak intensities. Most 2P microscopists therefore use femtosecond-pulsed lasers (which is currently patented by Zeiss). As photons move through a tissue, they are dispersed inversely proportional to pulse length (a phenomenon called group velocity dispersion [GVD] or chirp), mitigating the advantages of femtosecond versus picosecond excitation. This can be corrected by automatic compensation (termed "prechirp" or "negative chirp"), a feature now routinely offered by 2P laser manufacturers. Optimal laser power varies with depth of imaging, as does detector gain. Appropriate settings for deep tissues (e.g., high gain and laser power) result in detector saturation at shallower depths. To image throughout thick sections, some systems include software that modulates laser power and detector sensitivity with the depth of imaging, automatically correcting for the depth of the focal plane. This useful feature should be considered when selecting a system. Finally, although Ti:Sapphire lasers are tunable, they do not rapidly switch wavelengths during acquisition, limiting each imaging series to a single wavelength. To increase the number of colors that can be acquired simultaneously, the latest 2P microscopes can be equipped with two lasers, optimized for output at low versus high wavelengths. No doubt a good thing, but there are the inevitable costs both monetary (lasers are expensive, as are service contracts) and technical: (1) systems are more difficult to install and operate, (2) two lasers operating simultaneously cause twice the phototoxicity, (3) two lasers operating sequentially slow the acquisition rate two-fold, and (4) data file sizes increase with additional colors. Next decision: Will the laser light be delivered through an upright or inverted microscope (also a vexing decision for regular confocal microscopes). Each design has its advantages, as each is optimally suited for particular organs/tissues—the key word being optimal, since each can be adapted to image just about anything. For instance, mouse inguinal lymph nodes (LNs) are optimally imaged by an inverted microscope, while an upright microscope is better suited for popliteal LNs. Two key parameters are determined by microscope orientation: the lenses that can be used and the physical structures that can be imaged (e.g., mouse knees only bend one way). Dipping lenses (i.e., lenses designed to image while immersed in aqueous solutions) are difficult to employ with inverted microscopes, which is a significant disadvantage as dipping lenses typically feature the longest working distances (i.e., the longest distance from the objective that can be focused). While inverted scopes require less elaborate means of tissue immobilization (Sumen et al., 2004Sumen C. Mempel T.R. Mazo I.B. von Andrian U.H. Intravital microscopy: visualizing immunity in context.Immunity. 2004; 21: 315Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar) as the mouse is simply resting on glass placed on the objective, some organs are more easily imaged when the objective can just be placed on top of the organ. An objective inverter is commercially available (LSM TECH, Etters, PA) to convert an inverted microscope into an upright objective orientation, but at cost of light excitation and detection intensity. Of course, building a 2P microscope system requires far more knowledge and technical expertise than purchasing a turnkey system. The do-it-yourself advantages include (1) cost (homemade systems are usually far cheaper than turnkey systems); (2) optimized light path for vital imaging (turnkey systems are typically built to also perform standard LSCM, which requires compromises in the light path); and (3) hard-earned knowledge (by the time you are done, you will undoubtedly learn a great deal about customizing your microscope to your particular application). Data management is not to be taken lightly when embarking upon your 2PLSM adventure. A byproduct of the most exciting feature of 2PLSM—the generation of both 3D and 4D (the fourth dimension being time) data sets—is enormous file sizes. A typical data file for a 3D reconstruction of a 30 min imaging sequence is 500 MB. Given that 10–20 sequences will typically be generated in a single day, 100 gB of data can easily accumulate within weeks by a keen researcher. It is wise to devise a plan for data storage (and backup!) at the outset of MP experiments. All those bytes must be processed and analyzed. The most widely used software platforms for 2PLSM analysis are Imaris (Bitplane AG, Zurich, Switzerland) and Volocity (Improvision, Coventry, England). Each program has many useful features including (1) manipulation of large data sets, (2) filtering of data to remove background noise (often a reality due to low signal levels at increasing depths), (3) 3D rendering and movie generation, and (4) object tracking over time. Both Imaris and Volocity support user programming allowing the researcher to tailor the program to their needs, e.g., analysis of the time of contact between two cell types. These features allow statistical analysis of the behavior of cells under different experimental conditions (such as in the presence of pathogen-derived antigen or not). While these software platforms are superb, they are expensive and require high-performance computers with high-end graphics cards to work efficiently. Additionally, to generate appropriately annotated movies for presentation and publication (publication tip: journal reviewers love legends within movies!), video editing software is generally necessary (of course, the movie files themselves should not be manipulated!). OK, you're up to speed with 2PLSM hardware and data analysis software. Now for the rasion d'être, imaging host-pathogen interactions. This will require all of your expertise in your chosen experimental mouse infection system. The first decision is what organ to image. Many mouse tissues and sites have been imaged to date (Sumen et al., 2004Sumen C. Mempel T.R. Mazo I.B. von Andrian U.H. Intravital microscopy: visualizing immunity in context.Immunity. 2004; 21: 315Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). Pathogen-dependent organ tropism in combination with input dose and route of infection will determine which tissues are infected and the relevant immune organs that can be imaged (Figure 3). While you should (obviously) aim for the most-physiological route of infection and input infectious dose, this is not always feasible, particularly at the start. For example, small numbers of microorganisms located in a large organ (such as a liver) will frustrate imaging efforts since few, if any organisms will be located with the typical 300 μM working depth of the microscope. Choosing a physiological infectious dose is frequently hampered by ignorance of the details of natural pathogen transmission—e.g., the number of infectious virions transmitted by a mosquito bite (not necessarily a small number! [Styer et al., 2007Styer L.M. Kent K.A. Albright R.G. Bennett C.J. Kramer L.D. Bernard K.A. Mosquitoes inoculate high doses of West Nile virus as they probe and feed on live hosts.PLoS Pathog. 2007; 3: 1262-1270PubMed Google Scholar]). In common with many experimental systems, initial 2PLSM analysis of host-pathogen interactions typically uses relatively high numbers of pathogens delivered by hypodermic syringe either intravenously, in the skin, or directly to a relevant organ. With experience, the system can be made increasingly more physiological (and relevant) by lowering the dose and modifying the route of infection. As an example, Jin et al., 2007Jin Y. Kebaier C. Vanderberg J. Direct microscopic quantification of dynamics of Plasmodium berghei sporozoite transmission from mosquitoes to mice.Infect. Immun. 2007; 75: 5532-5539Crossref PubMed Scopus (70) Google Scholar used traditional fluorescence microscopy to image the delivery of malaria parasites to a mouse ear by the bite of an infected mosquito. One of the most critical and challenging aspects of 2PLSM imaging is optimizing fluorescent molecules for imaging. Bacteria and parasites can usually be genetically engineered to constitutively express sufficient copies of a fluorescent protein (FP) to enable direct visualization of the pathogen in tissues. This allows direct observation of the input pathogen itself and also its progeny. Viruses are typically more challenging to label. A few large viruses can potentially be engineered to express a fluorescent structural protein that is incorporated in virions in sufficient copy number to enable direct visualization of virions. Most viruses, however, probably cannot accommodate such a drastic structural change. While viruses can be directly labeled using fluorescent membrane (for enveloped viruses) or protein dyes, labeling can drastically reduce infectivity and, in any event, will enable visualization only of input virus and will provide limited (if any) information regarding the expression of viral gene products in host cells. No matter the virion labeling strategy, nonfilamentous viruses are smaller than the resolution of 2PLS microscopes and will be visualized as geometric points. Virus-based 2PLSM studies will typically utilize viruses that encode FPs synthesized by infected cells but excluded from virions. In general, as strong a promoter as possible should be chosen to express the protein, with the caveat that foreign gene expression should not interfere with the infectious cycle. Greater fluorescent reporter expression will maximize detection of cells that are "weakly" infected, facilitate detection of infected cell extensions (e.g., detection of cytoplasmic or membrane bound FPs in dendritic cell processes), and improve image quality and depth of detection. Considerable thought should be given to the form of antigen expressed. If pathogen interaction with T cells is a goal of the study, the FP can be fused with sequences that encode one or more determinants recognized by MHC class I- or class II- restricted TCR-transgenic mice. While most fluorophores used in 2PSLM result in cytoplasmic staining of the cell of interest, targeting of the FP to various cellular organelles has advantages/disadvantages. Nuclear targeting concentrates the FP in a relatively small volume, increasing the sensitivity of detection and providing clear demonstration that cells are truly infected and have not endocytosed FP from other infected cells. Membrane targeting enables clear delineation of infected cell borders and facilitates observations of cell-surface interactions between infected cells and responding immune cells, but can be weak in comparison to nuclear/cytoplasmic staining. Mitochondrial targeting is of great use if detecting virus-induced apoptosis is a central goal of the study. Similarly, in viral systems with temporally controlled gene expression, it may be possible to kinetically control virus-driven FP expression (by placement after a regulated promoter). Generally, expression throughout the infectious cycle will probably be easiest for the identification of infected cells. As confidence in the system grows, it is possible to refine the experiments by temporally limiting FP expression to early or late parts of the infectious cycle. Some viruses cannot be engineered to express FPs while maintaining full infectivity, because their genomes cannot accept additional genetic information without replacing essential genes (e.g., influenza A virus). For pathogens that fall into this dreaded category, an alternative (as-yet-unreported) strategy is to rely on virus-induced transactivation to induce FP expression in host cells. For instance, a very early event in most viral infections is the production of type I interferons. Transgenic mice that express a FP under control of an interferon-responsive promoter should enable detection of local infection, with the obvious caveat that neighboring cells exposed to interferons will also express the FP. Promoter leakiness could also wreak havoc on such a system. The discovery of more specific promoters should facilitate the identification of infected cells using this strategy. Once you have arrived upon a suitable method of labeling your pathogen, you must decide which FP to use. The standard FP in 2PLSM (and all) imaging is eGFP. eGFP is a safe choice for initial studies, as it is brighter and more photostable than many alternative FPs and has been used by many researchers who can offer advice. A close relative of eGFP, Venus-eYFP, is a bright alternative in the green/yellow range (Giepmans et al., 2006Giepmans B.N. Adams S.R. Ellisman M.H. Tsien R.Y. The fluorescent toolbox for assessing protein location and function.Science. 2006; 312: 217-224Crossref PubMed Scopus (2315) Google Scholar). Cyan FP (eCFP) has been used for imaging, but has a lower quantum yield (i.e., is less bright) than green/yellow FPs. Red FPs (such as dsRed, tdTomato, and mCherry) have also been utilized, but are more difficult to excite using 2P, have lower quantum yields, and may photobleach rapidly. Although a far red FP now exists (mPlum), it has been difficult to excite using standard 2P lasers. The principal reason for turning to eGFP alternatives for pathogen tagging is the growing number of eGFP/eYFP-expressing transgenic mice that can be used to image host responses to infection in combination with blue or red pathogens. FP-expressing pathogens used in intravital imaging studies reported to date are listed in Table 1.Table 1Pathogens Studied in flagrante delictoPathogenFluorophoreCommentsReferenceVirusesVacciniaeGFPStatic image of infection using 2PLSM(Norbury et al., 2002Norbury C.C. Malide D. Gibbs J.S. Bennink J.R. Yewdell J.W. Visualizing priming of virus-specific CD8+ T cells by infected dendritic cells in vivo.Nat. Immunol. 2002; 3: 265-271Crossref PubMed Scopus (287) Google Scholar)VacciniaeGFP2PLSM movies of lymph node infection(Hickman et al., 2008Hickman H.D. Takeda K. Skon C.N. Murray F.R. Hensley S.E. Loomis J. Barber G.N. Bennink J.R. Yewdell J.W. Direct priming of antiviral CD8+ T cells in the peripheral interfollicular region of lymph nodes.Nat. Immunol. 2008; 9: 155-165Crossref PubMed Scopus (199) Google Scholar)Vesicular stomatitseGFP2PLSM movies of lymph node infection(Hickman et al., 2008Hickman H.D. Takeda K. Skon C.N. Murray F.R. Hensley S.E. Loomis J. Barber G.N. Bennink J.R. Yewdell J.W. Direct priming of antiviral CD8+ T cells in the peripheral interfollicular region of lymph nodes.Nat. Immunol. 2008; 9: 155-165Crossref PubMed Scopus (199) Google Scholar)UV-VSVAlexaFluor-568/4882PLSM movies of inactivated virions in the lymph node(Junt et al., 2007Junt T. Moseman E.A. Iannacone M. Massberg S. Lang P.A. Boes M. Fink K. Henrickson S.E. Shayakhmetov D.M. Di Paolo N.C. et al.Subcapsular sinus macrophages in lymph nodes clear lymph-borne viruses and present them to antiviral B cells.Nature. 2007; 450: 110-114Crossref PubMed Scopus (627) Google Scholar)ProkaryotesSalmonella typhimuriumDsRed2PLSM movies of mucosal DC response(Chieppa et al., 2006Chieppa M. Rescigno M. Huang A.Y. Germain R.N. Dynamic imaging of dendritic cell extension into the small bowel lumen in response to epithelial cell TLR engagement.J. Exp. Med. 2006; 203: 2841-2852Crossref PubMed Scopus (577) Google Scholar)Mycobacterium boviseGFP or DsRed2PLSM movies of liver infection(Egen et al., 2008Egen J.G. Rothfuchs A.G. Feng C.G. Winter N. Sher A. Germain R.N. Macrophage and T cell dynamics during the development and disintegration of mycobacterial granulomas.Immunity. 2008; 28: 271-284Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar)Escherichia coliGFP2PLSM movies of kidney tubule infection(Mansson et al., 2007Mansson L.E. Melican K. Boekel J. Sandoval R.M. Hautefort I. Tanner G.A. Molitoris B.A. Richter-Dahlfors A. Real-time studies of the progression of bacterial infections and immediate tissue responses in live animals.Cell. Microbiol. 2007; 9: 413-424Crossref PubMed Scopus (76) Google Scholar)Borrelia burgdorferiGFP2PLSM movies of spirochetes in skin microvasculature(Moriarty et al., 2008Moriarty T.J. Norman M.U. Colarusso P. Bankhead T. Kubes P. Chaconas G. Real-time high resolution 3D imaging of the lyme disease spirochete adhering to and escaping from the vasculature of a living host.PLoS Pathog. 2008; 4: e1000090Crossref PubMed Scopus (149) Google Scholar)EukaryotesPlasmodium bergheiRedStarIntravital movies of parasites in the liver(Frevert et al., 2005Frevert U. Engelmann S. Zougbede S. Stange J. Ng B. Matuschewski K. Liebes L. Yee H. Intravital observation of Plasmodium berghei sporozoite infection of the liver.PLoS Biol. 2005; 3: e192Crossref PubMed Scopus (261) Google Scholar)Plasmodium yoeliiGFP (GFPmut3)Intravital movies of parasite late liver stages(Tarun et al., 2006Tarun A.S. Baer K. Dumpit R.F. Gray S. Lejarcegui N. Frevert U. Kappe S.H. Quantitative isolation and in vivo imaging of malaria parasite liver stages.Int. J. Parasitol. 2006; 36: 1283-1293Crossref PubMed Scopus (93) Google Scholar)Plasmodium yoeliiGFPIntravital movies of parasite release from the liver(Baer et al., 2007Baer K. Klotz C. Kappe S.H. Schnieder T. Frevert U. Release of hepatic Plasmodium yoelii merozoites into the pulmonary microvasculature.PLoS Pathog. 2007; 3: e171Crossref PubMed Scopus (156) Google Scholar)Leishmania majorRFP2PLSM movies of parasites in the skin(Peters et al., 2008Peters N.C. Egen J.G. Secundino N. Debrabant A. Kimblin N. Kamhawi S. Lawyer P. Fay M.P. Germain R.N. Sacks D. In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies.Science. 2008; 321: 970-974Crossref PubMed Scopus (598) Google Scholar)Toxoplasma gondiitdTomato2PLSM movies of parasites in the lymph node(Chtanova et al., 2008Chtanova T. Schaeffer M. Han S.J. van Dooren G.G. Nollmann M. Herzmark P. Chan S.W. Satija H. Camfield K. Aaron H. et al.Dynamics of neutrophil migration in lymph nodes during infection.Immunity. 2008; 29: 487-496Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar) Open table in a new tab Now that you can "see" your pathogen, what about the host response? The easiest and often the most physiological way to image the host cellular immune response is to use transgenic mice expressing FPs labeling specific cell populations. The most commonly used mice for immunological 2PLSM studies (and some that will undoubtedly soon be used) are listed in Table 2. Most of the mice express eGFP, necessitating the use of an alternate FP for pathogen expression (note, however, that while suboptimal, it is possible to use a pathogen also expressing eGFP). In some cases, weak transgene expression will significantly limit imaging.Table 2FP-Expressing Transgenic MiceCD11c-DTR-eGFPDCWeak cell surface eGFP(Jung et al., 2002Jung S. Unutmaz D. Wong P. Sano G. De los S.K. Sparwasser T. Wu S. Vuthoori S. Ko K. Zavala F. et al.In vivo depletion of CD11c(+) dendritic cells abrogates priming of CD8(+) T cells by exogenous cell-associated antigens.Immunity. 2002; 17: 211-220Abstract Full Text Full Text PDF PubMed Scopus (1440) Google Scholar)CD11c-eYFPDCVenus eYFP(Lindquist et al., 2004Lindquist R.L. Shakhar G. Dudziak D. Wardemann H. Eisenreich T. Dustin M.L. Nussenzweig M.C. Visualizing dendritic cell networks in vivo.Nat. Immunol. 2004; 5: 1243-1250Crossref PubMed Scopus (657) Google Scholar)CCL17-GFPLymph node DCGFP(Alferink et al., 2003Alferink J. Lieberam I. Reindl W. Behrens A. Weiss S. Huser N. Gerauer K. Ross R. Reske-Kunz A.B. Ahmad-Nejad P. et al.Compartmentalized production of CCL17 in vivo: strong inducibility in peripheral dendritic cells contrasts selective absence from the spleen.J. Exp. Med. 2003; 197: 585-599Crossref PubMed Scopus (144) Google Scholar)Yet40DC, macrophageseYFP(Reinhardt et al., 2006Reinhardt R.L. Hong S. Kang S.J. Wang Z.E. Locksley R.M. Visualization of IL-12/23p40 in vivo reveals immunostimulatory dendritic cell migrants that promote Th1 differentiation.J. Immunol. 2006; 177: 1618-1627Crossref PubMed Scopus (94) Google Scholar)MHC II-eGFPDC, B cells (intermediate), macrophages (dim)eGFP(Boes et al., 2002Boes M. Cerny J. Massol R. Op den Brouw M. Kirchhausen T. Chen J. Ploegh H.L. T-cell engagement of dendritic cells rapidly rearranges MHC class II transport.Nature. 2002; 418: 983-988Crossref PubMed Scopus (334) Google Scholar)CD11b-DTR-eGFPMacrophagesWeak cell surface eGFP(Cailhier et