The authors gratefully acknowledge funding by the DFG Collaborative Research Centers SFB 1181 and TRR 241. The Optical Imaging Centre Erlangen (OICE) and in particular Ralf Palmisano, Philipp Tripal and Tina Fraa&#223; (Project Z2 of the DFG CRC 1181) are acknowledged for expert technical support for light-sheet fluorescence microscopic imaging.

Experiments involving animals were performed in accordance with protocols approved by the relevant local authorities in Erlangen (Regierung von Unterfranken, W&#252;rzburg, Germany). Mice were housed under specific pathogen-free conditions. The collection of human blood was approved by the local ethical committee and the institutional review board of the University of Erlangen-Nuremberg. Each patient gave written informed consent.
1. Induce Allergic Lung Inflammation in Mice
<p class="jove_c...

The here described experimental setting provides the opportunity to monitor the lung homing capacity of primary human immune cells under in vivo inflammatory conditions and thereby relevantly complements classically performed in vitro adhesion and chemotaxis assays. To take into account specific anatomic organ characteristics of the lung, important aspects of immune cell homing (including chemotaxis and cell distribution within the target organ) as well as the clinical relevance and transfera...

Classic inflammatory disorders of the lung, such as allergic asthma and chronic obstructive pulmonary disease (COPD), are well known to be driven by an increased recruitment of activated lymphocytes into the pulmonary tissue1,2. Lymphocyte-released cytokines (e.g., IL-4, IL-5, IL-9, IL-13, IFN-&#947; and TNF-&#945;) further promote chemotaxis of innate and adaptive immune cells, induce fibrotic airway remodeling or directly damage the lung parenchyma2. So far, the underlying mechanisms responsible for the pathological accumulation of lymphocytes within lung tissue are not yet fully understood. In analogy to tissue-selective T cell imprinting described for gut and skin homing, pulmonary dendritic cells (DCs) are obviously able to prime peripheral T cells for preferential lung infiltration, at least partly via the induction of CCR4 expression on the surface of lymphocytes3. Besides CCR4, airway-infiltrating T cells are also characterized by a particularly increased expression of the chemokine receptors CCR5 and CXCR3 compared to T cells within the peripheral blood1,4,5. Overall, existing data are consistent with the concept that lung homing of T lymphocytes under physiological or inflammatory conditions involves a number of different chemokine receptors and their respective ligands and thus crucially depends on a closely controlled collaboration between innate and adaptive immune cells1. Especially, during the initial phase of pathogen or allergen exposure, cells of the innate immune system respond to TLR stimulation or to IgE-mediated cross-linking by the immediate release of different chemoattractants, like LTB4, CCL1, CCL17, CCL22, CCL20, CXCL10 and PGD21,6,7. As a prime example, the interaction between PGD2 and the chemoattractant receptor CRTh2 is known to be of particular importance for chemotaxis of Th2 cells and thus appeared as promising therapeutic target in the clinical management of asthma. Indeed, patients with moderate asthma showed an improvement of symptoms and a significant increase of the forced expiratory volume in one second (FEV1) after treatment with a selective CRTh2 antagonist compared to the placebo group8,9. In a more progressed state of the inflammatory response, already recruited T cells are able to further amplify pulmonary lymphocyte accumulation via the release of IL-4 and IL-13 as potent stimuli for pulmonary DCs. Subsequently, these myeloid-derived innate cells up-regulate the expression of CCL17 and CCL22 in a STAT6-dependent manner1,10,11.&#160; Although the complexity of the described scenario still hinders a complete understanding of T cell lung homing, it offers a plethora of molecular targets for a potentially optimized therapeutic control of inflammatory or allergic pulmonary diseases. Therefore, there is an urgent need of innovative experimental techniques, which are able to further deepen and complement our knowledge in the field of T cell chemotaxis and lung homing.
Due to the fact that lung homing of lymphocytes within the human body is influenced by multiple cellular, humoral and physical parameters1, most of the existing experimental methods are not able to model the whole complexity of this immunological process. Instead, many standard protocols for the analysis of lung homing selectively focus on a specific aspect involved in the cascade of lymphocyte attraction, adhesion, migration and retention. Besides a purely descriptive determination of the mRNA or protein expression pattern of integrins and chemokine receptors on peripheral or lung-infiltrating lymphocytes and the complementary measurement of respective chemokine levels in blood, bronchoalveolar lavage (BAL) or pulmonary tissue12,13,14,15, well-established in vitro cell culture assays allow a functional characterization of lymphocyte adhesion or chemotaxis upon defined experimental conditions16,17,18. In principle, static in vitro adhesion assays monitor the binding capacity of cultured lymphocytes to an endothelial monolayer or to glass slides coated with recombinant endothelial adhesion molecules (e.g., MAdCAM-1, VCAM-1), while standard in vitro chemotaxis assays are usually applied in order to quantify the ability of lymphocytes to migrate along a chemokine gradient in a transwell system19. Both in vitro settings enable a controlled adjustment and modulation of experimental conditions, but on the other hand lack important variables known to critically impact on in vivo chemotaxis and adhesion of lymphocytes. Predominantly, static cell culture assays disregard the influence of shear forces caused by the permanent blood flow19 and potentially neglect the involvement of the surrounding immunological milieu and interacting non-lymphocyte immune cells, both present in a living organism. In order to overcome these limitations, the interpretation of results acquired in static in vitro chemotaxis or adherence assays need further validation in dynamic adhesion experiments under flow conditions20,21 and in in vivo models of inflammatory organ pathology19. Indeed, important conclusions on the regulation of T cell lung homing under inflammatory or allergic conditions could be drawn from animal studies analyzing genetically modified mice in defined models of different pulmonary diseases3,22,23. The quantitative comparison of lung infiltrating lymphocytes between wildtype mice and mice with a deficiency for a specific gene of interest represents a well-established and broadly used tool for defining the impact of particular cellular pathways or receptors on the disease-driven pattern of T cell distribution. However, in contrast to before discussed in vitro cell culture assays, a study design based on classical animal models lacks the ability to analyze and monitor primary human T cells directly derived from the blood or BAL of patients suffering from an inflammatory lung disease. Thus, it still remains challenging to functionally validate whether a diagnostically specified lung disease is able to imprint human lymphocytes for preferential lung tropism and how far clinical parameters might impact on this scenario. Recently, a very elegant in vivo approach was introduced in the context of inflammatory bowel diseases (IBD), which was able to overcome most of these limitations and opened new avenues for advanced translational studies on intestinal lymphocyte homing24. Taking advantage of protocols for solvent-based tissue clearing followed by cross-sectional light-sheet fluorescence microscopy as a powerful imaging tool, it was possible to visualize the infiltration and distribution of adoptively transferred human T cells in the intestine of colitic immunodeficient mice24. In particular, this experimental setting implemented two main innovations: (1) Primary human immune cells can be analyzed under experimentally defined in vivo conditions; (2) a rather large area of the diseased organ (about 1.5&#160;cm&#160;x&#160;1.5&#160;cm) can be imaged in high resolution quality, followed by 3D-reconstruction. Moreover, several recent studies successfully established the use of solvent-based tissue clearing and light-sheet fluorescence microscopy as important tools for advanced lung imaging25,26. In order to benefit from this technological progress in the field of pulmonary immunology, we now adopted the system for analysis of lung homing.
The here presented protocol provides a step-by-step introduction how to purify and fluorescently label primary human T cells for transfer into mice with induced pulmonary inflammation and, moreover, describes in detail the subsequent process of light-sheet fluorescence microscopic imaging, including organ preparation and image processing. Overall, we hope to support future translational studies in the field of inflammatory or allergic lung diseases by introducing a sophisticated, but nevertheless feasible, experimental model for monitoring human lymphocyte lung homing upon in vivo conditions.

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			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
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			<td height="20" style="height:20px;">CD4 MicroBeads, human</td>
			<td>Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany</td>
			<td>130-045-101</td>
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			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
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			<td height="20" style="height:20px;">FBS (fetal bovine serum) Good Forte</td>
			<td>PAN-Biotech GmbH, Aidenbach, Germany</td>
			<td>P40-47500</td>
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			<td height="20" style="height:20px;">Filter 100 &#181;m&#160;</td>
			<td>VWR International Germany GmbH, Darmstadt, Germany</td>
			<td>732-2758</td>
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			<td height="20" style="height:20px;">Imaris Image Analysis Software 9.0.2</td>
			<td>Bitplane AG, Zurich, Switzerland</td>
			<td>n.a.</td>
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			<td>Abberior Instruments GmbH, G&#246;ttingen, Germany</td>
			<td>n.a.</td>
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			<td height="20" style="height:20px;">Ketamin&#160;</td>
			<td>Inresa Arzneimittel GmbH, Freiburg, Germany</td>
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			<td height="20" style="height:20px;">LaVision UltraMicroscope II</td>
			<td>LaVision BioTec GmbH, Bielefeld, Germany</td>
			<td>n.a.</td>
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			<td height="20" style="height:20px;">MACS MultiStand</td>
			<td>Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany</td>
			<td>130-042-303</td>
			<td></td>
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			<td height="20" style="height:20px;">Multifly cannula 20 G</td>
			<td>Sarstedt AG &#38; Co., N&#252;mbrecht, Germany</td>
			<td>851638035</td>
			<td></td>
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			<td height="20" style="height:20px;">30 G needle</td>
			<td>B. Braun Melsungen AG, Melsungen, Hessen, Germany</td>
			<td>9161502</td>
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			<td>neoLab Migge GmbH, Heidelberg, Germany</td>
			<td>C-1003</td>
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			<td height="20" style="height:20px;">Pattex Glue</td>
			<td>Henkel AG &#38; Co, D&#252;sseldorf, Germany</td>
			<td>PSK1C</td>
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			<td>Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany</td>
			<td>130-042-401</td>
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			<td height="21" style="height:21px;">Tube 1.5 ml</td>
			<td>Sarstedt AG &#38; Co., N&#252;mbrecht, Germany</td>
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			<td height="20" style="height:20px;">Tube 2 ml</td>
			<td>Sarstedt AG &#38; Co., N&#252;mbrecht, Germany</td>
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			<td height="20" style="height:20px;">Tube 2 ml, brown</td>
			<td>Sarstedt AG &#38; Co., N&#252;mbrecht, Germany</td>
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			<td height="20" style="height:20px;">Tube 15 ml</td>
			<td>Sarstedt AG &#38; Co., N&#252;mbrecht, Germany</td>
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			<td height="20" style="height:20px;">Tube 50 ml</td>
			<td>Sarstedt AG &#38; Co., N&#252;mbrecht, Germany</td>
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advanced imaging of lung homing human lymphocytes in an experimental in vivo model of allergic inflammation based on light-sheet microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The presented protocol describes an experimental mouse model, which allows monitoring and quantifying the accumulation of adoptively transferred human T lymphocytes in the lung via light-sheet fluorescence microscopy.&#160;Figure 1A provides a schematic overview of the in vivo steps of the experimental schedule. In order to guarantee reliable results, it is of substantial importance to ensure a good quality of the isolated and fluorescently labeled human CD4+ T ce...

Watch this Scientific Journal Video about Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy at JoVE.com

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an ...

Our technique allows the precise imaging of lung-accumulated human immune cells in in vivo inflammatory conditions providing exciting insights into the process of T cell lung homing. This protocol supports translational research on T cell lung homing and may thereby help in the identification of new targets for an optimized therapy of inflammatory or allergic pulmonary diseases. In particular, the ability to consider the influence of disease-specific immune cell properties, pulmonary tissue organization, and inflammatory milieu on T cell lung homing makes this method highly advantageous.Overall, our technique is of high value for immunological research in the field of chronic inflammatory lung diseases, which are often driven by an overwhelming tissue accumulation of activated immune cells. After confirming a lack of response to toe pinch in an at least 16-gram, six-week-old, anesthetized C57 black 6J mouse, use a micropipet to slowly deliver 10 microliters of freshly prepared papain solution into one nostril of the mouse once a day for three consecutive days. The day after the last papain administration, layer 10 milliliters of Ficoll medium under human peripheral blood from a healthy volunteer pre-diluted at a one to two ratio in PBS for density gradient separation by centrifugation.Transfer the peripheral mononuclear blood cell or PBMC interphase layer into a new 50-milliliter tube and collect the cells by centrifugation. Next, perform magnetic microbead isolation of CD4 positive cells according to the manufacturer's protocol. Re-suspending the bead isolated CD4 positive T cells at an up to one times ten to the seventh T cells per at least 500 microliters of PBS concentration.Label the cells with an appropriate red light excitable cell proliferation dye for 10 minutes at 37 degrees Celsius. At the end of the incubation, arrest the labeling reaction with five volumes of RPMI medium supplemented with 10%FBS for five minutes on ice followed by three washes in medium. For adoptive transfer of the human CD4 positive T cells into papain-exposed recipient mice, re-suspend the fluorescently labeled T cells to a one times 10 to the seventh cells per milliliter concentration in PBS and load 100 microliters of cells into one one-milliliter syringe equipped with a 30-gauge needle per recipient animal, then inject the entire volume of cells from one syringe into the tail vein a papain-treated animal maintaining the needle tip within the vein for an additional five seconds after cell delivery to prevent discharge of the cell suspension.Three hours after adoptive cell transfer, puncture the right ventricle of each euthanized mouse with a 21-gauge cannula connected to a catheter and make an incision in the left ventricle. Slowly perfuse the lung with 20 milliliters of ice-cold PBS supplemented with five millimolar EDTA followed by two milliliters of ice-cold 4%paraformaldehyde. When all of the fixative has been flushed through the tissue, remove the salivary glands and cut the sternal hyoid muscle to allow a forceps to be slid under the trachea to expose the airtube tissue.Perform an initial puncture of the exposed trachea with a 30-gauge needle before replacing the needle with a blunt 30-gauge catheter to prevent further damage to the tissue. Use a needle holder to seal the junction between the inserted catheter and the trachea, and carefully fill the airways with approximately 37 degrees Celsius 0.75%agarose until complete unfolding of the lung. A tight junction between catheter and trachea is critical for expanding the lung to its physiological size and maintaining tissue architecture for imaging.When the agarose has completely solidified, remove the catheter and carefully transfer the lung into a darkened, two-milliliter tube filled with 4%paraformaldehdye. Next, dehydrate the tissue with sequential ethanol incubations under continuous rotation at 31 rotations per minute and four degrees Celsius as indicated for four hours per incubation. After complete dehydration, transfer the sample into ethyl cinnamate for an overnight incubation at room temperature until the tissue appears translucent.For a light-sheet fluorescent microscopy, use a small drop of organic solvent stable glue to stick the lung to the sample holder of the microscope and set the lung lobe onto the holder in an upright position. Place the sample holder into its chamber and select the appropriate lasers for the experiment in the instrument software. Next, use the sliders under Optics to set a sheet width that numerical aperture that uniformly reveal the whole organ or a specific section of interest.Use the slider to set the laser intensity under laser transmission control for each selected laser and click Apply. Use Advanced measurement settings to merge the left and right light sheets to create a homogeneously illuminated image. Define the start and end positions of the Z stack to be measured under scan range and set the step size to five micrometers then select autosave to save the files automatically and begin the measurement to capture the images.When all of the images have been acquired, activate the Surpass button in the 3D post-imaging analysis software and load the first image from one Z stack to initiate the opening and automatic 3D reconstruction of all other images of the selected Z stack, then under Display Adjustment, adjust the intensity, black level and contrast for each channel. To compare the accumulation of human cells within the pulmonary tissue across different samples, select Edit and Crop 3D to define a lung cube with a specific volume. To quantify the labeled cells within the defined lung cube, open Add New Spots in the toolbar, click Skip automatic creation, edit manually, and select the 640 nanometer channel.Set the radius scale to 10.00 microns, click Select, and under Edit, Shift click with the left mouse button to individually mark each fluorescing cell with a dot. The overall number of counted cells will be displayed in the statistics. To capture an image, use the snapshot tool and or use the animation tool to capture a video.Microbead-based cell enrichment and subsequent fluorescence labeling as demonstrated typically result in a CD4 positive purity of at least 95%and a successful labeling of these cells as determined by flow cytometry. Ethyl cinnamate-based tissue clearing achieves a high level of organ transparency indicating a successful refractive index matching. The auto-fluorescent signal of the lung tissue offers a helpful tool to image the anatomic structure of the lung by light-sheet microscopy which can be displayed as a mean intensity projection or in surface mode.Compared to conventional microscopy, light-sheet microscopy offers the advantage of whole organ imaging and subsequent 3D reconstruction allowing the identification and visualization of proliferation dye-labeled, transferred human T cells in the context of the whole organ, moreover, the preference of transferred human T cells for selective accumulation within the inflamed lung tissue of papain-exposed animals is further supported by the fact that human CD4 positive T cells cannot be retrieved from the intestinal mucosa of the recipient animals. For the quantification and comparison of the pulmonary accumulation of human CD4 positive T cells between different conditions, the labeled cells can also be counted in several lung cubes of defined volume as demonstrated. Careful tissue preparation including lung perfusion, inflation and tissue clearing is highly important for an optimized generation of high resolution light-sheet microscopic images.To further confirm the successful diapedesis of transferred human lymphocytes, this protocol can easily be combined with the flow cytometric quantification of human lymphocytes within the murine bronchoalveolar lavage. The ability to monitor immune cells derived from individual patients in in vivo conditions supports the identification of functional alterations in immune cells imprinted by a particular disease or therapy.

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7.5K ビュー.  University Hospital of Erlangen. 我々の技術は、インビボ炎症状態における肺蓄積ヒト免疫細胞の正確なイメージングを可能にし、T細胞肺ホーミングのプロセスに関するエキサイティングな洞察を提供する。このプロトコルは、T細胞肺ホーミングに関する翻訳研究をサポートし、それによって炎症性またはアレルギー性肺疾患の最適化された治療のための新しい標的の同定に役立つかもしれない。特に...