We are grateful to the personnel of the Biomedical Services Unit of the University of Birmingham for taking care of our animal colonies. This work was supported by the EU FP7 integrated project INFLACARE to JC.

Mice were bred and maintained under SPF conditions in the Biomedical Service Unit at the University of Birmingham according to UK Home Office and local ethics committee regulations. All procedures described in this protocol are covered under a Project License approved by both local ethics committee and the Home Office.
1. Isolation of Newborn LNs
<ol>
	<li>Sacrifice the newborn mice by cervical dislocation.</li>
	<li>Section the head, and open the body with scissors from the top of the thoracic region to the bottom of the abdominal region and remove carefully all the viscera (heart, lungs, liver, intestine, kidney, bladder) from the abdominal cavity.</li>
	<li>Transfer the body into a 90 mm Petri dish containing RF10 media (RPMI1640 media supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM L-glutamine). From this step onwards, the tissues will be kept under sterile conditions and handled in a tissue culture hood.</li>
	<li>Transfer the body into a new 90 mm Petri dish containing RF10.</li>
	<li>Under the dissecting microscope, detach carefully the peritoneum from the skin in the inguinal region. The inguinal LN is situated at the intersection of three blood vessels in the fat pad.</li>
	<li>Remove carefully the LN. Make sure that all adipose tissue is removed. Transfer the LN into a 50 mm Petri dish containing RF10 media. Keep the tissues on ice while continuing with the procedure.</li>
</ol>
2. Isolation of E18.5 Fat Pads
<ol>
	<li>To generate mouse embryos at day 18.5 of gestation (E18.5), set up timed pregnancies by placing a female and a male mouse per cage overnight. Separate the mice in the morning and check for vaginal plugs (gestational day E0.5).
	<ol>
		<li>Euthanize the plugged females at day 18.5 of pregnancy by cervical dislocation.</li>
		<li>Remove the embryos and placed them in a 90 mm Petri Dish containing RF10.</li>
	</ol>
	</li>
	<li>From this step onwards, tissues should be handled using sterile technique in the tissue culture hood. Under the dissecting microscope, section the head, open the embryos with scissors from the thoracic region to the bottom of the abdominal region and remove carefully all the viscera (heart, lungs, liver, intestine, kidney, bladder) from the body cavity.</li>
	<li>Clean the body from all remaining visceral tissues and wash it in PBS to eliminate all traces of blood that could make the dissection more difficult.</li>
	<li>Transfer the clean bodies into a fresh 90 mm Petri dish containing RF10.</li>
	<li>Carefully detach the peritoneum from the skin in the inguinal region. The inguinal LN is situated at the intersection of three blood vessels in the fat pad.</li>
	<li>Carefully remove the LN and discard it. It is very important to completely remove the LN to ensure that the fat pad used for the chimeras is not contaminated by any lymphoid stromal cells.</li>
	<li>Remove the inguinal fat pad and transfer it into a 50 mm Petri dish containing RF10 media. Keep the tissues on ice while continuing with the procedure.</li>
</ol>
3. Generation of the LN-fat Pad Chimera
<ol>
	<li>Prepare the in vitro organ culture system15.
	<ol>
		<li>Sponge and filters preparation: This can be done in advance and sponges and filters stored sterile in a Petri dish in the culture hood.
		<ol>
			<li>Cut the Vulkan Underwrap in 1-1.5 cm2 pieces</li>
			<li>Boil the sponges for 2 hr in distilled water.</li>
			<li>Let the sponges dry for a couple of hours in the cell culture hood.</li>
			<li>Boil the filters for 20 min.</li>
			<li>Let the filters dry for a couple of hours in the cell culture hood.</li>
		</ol>
		</li>
		<li>Prepare DMEM media complemented with 10% fetal bovine serum, 10 mM HEPES, 1x&#160;MEM Nonessential Amino Acid Solution, 50 mM 2-Mercaptoethanol, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM L-Glutamine.</li>
		<li>Add 2 ml of media in a 50 mm Petri dish.</li>
		<li>Arrange one sponge at the bottom of the Petri dish. Wet both sides of the sponge by submerging them in the media.</li>
		<li>Place one filter on top of the sponge. The filter is positioned at the liquid-air interface.</li>
	</ol>
	</li>
	<li>Under the dissecting microscope, carefully reassociate one newborn LN with one embryonic fat pad.</li>
	<li>Place the LN-fat pad chimera on top of the filter.</li>
	<li>Transfer the Petri dish in a rectangular plastic box with a couple of holes on the lid and containing water at the bottom to ensure humidity.</li>
	<li>Seal the lid to the box with tape. Leave the two holes in the lid open.</li>
	<li>Transfer the box to a cell culture incubator at 37 &#176;C with 5% CO2. Allow the box to equilibrate for 2 hr, before sealing the holes in the lid with tape.</li>
	<li>Incubate the tissues for at least 2 days to allow the LN to attach to the fat pad and be transferable under the kidney capsule.</li>
</ol>
4. Transfer of the LN-fat Pad Chimera Under the Kidney Capsule of Host Mice
<ol>
	<li>Collect the LN-fat pad chimeras from the filter and transfer them under the kidney capsule of host mice. The kidney capsule transfer method has already been described 16.</li>
</ol>
5. Isolation of the Transferred LNs
<ol>
	<li>After 3-4 weeks under the kidney capsule, the LN-fat pads chimeras are ready to be harvested. Euthanize the host mice using approved guidance.</li>
	<li>Remove the kidney from the host mice.</li>
	<li>Under the dissecting microscope, isolate the LN-fat pad chimera and dissect out the LN.</li>
</ol>
6. Enzymatic Digestion of the Transferred LNs for Flow-Cytometric Analysis
<ol>
	<li>Make one incision in the LN with small scissors to allow the enzymes to penetrate the organ and facilitate the digestion.</li>
	<li>Place one LN in 1.5 ml Eppendorf containing 600 &#956;l of digestion buffer (RPMI 1% FCS, 2.5 mg/ml Collagenase D, 100 &#956;g/ml DNase I).</li>
	<li>Incubate for 30 min at 37 &#176;C on an agitating thermal block. Pipette up and down to help dissociation of tissues every 10 min.</li>
	<li>Add 6 &#956;l EDTA 0.5 M to the tube and pipette up and down to finish the dissociation of the tissues.</li>
	<li>Centrifuge for 5 min at 490 x g.</li>
	<li>Resuspend the cell pellet in the staining solution (PBS, 0.1% BSA, 5% rat serum and 5% mouse serum) containing the combination of primary antibodies.</li>
	<li>Incubate for 30 min on ice.</li>
	<li>Wash twice in PBS</li>
	<li>Incubate with secondary antibodies for 20 min if required.</li>
	<li>Wash 2x in PBS.</li>
	<li>Analyze on flow-cytometer.</li>
</ol>
7. Analysis of the Transferred LNs by Immunofluorescence
<ol>
	<li>Fixation for EYFP conservation during the freezing and cryosections process: Place the LN in a solution of PBS, 4% PFA and 10% sucrose and leave for 3-4 hr.</li>
	<li>Put a single small drop of cold O.C.T. compound on a small piece of aluminum foil. Avoid air bubbles. Carefully place the LN without any liquid in the middle of the drop.</li>
	<li>Freeze the organ by placing the aluminum foil on dry ice.</li>
	<li>Cut 8-10 &#956;m sections onto extra adhesives glass slides using a cryostat. Let the slides dry for 2 hr before storing them at -20 &#176;C.</li>
	<li>Staining procedures can vary according to primary antibodies used and can be optimized from the following protocol. Stain the sections with staining solution (PBS, 1% BSA) containing the combination of primary antibodies.</li>
	<li>Incubate for 1 hr at room temperature in a humidified chamber.</li>
	<li>Wash twice in PBS for 5 min</li>
	<li>Incubate with secondary antibodies for 1 hr.</li>
	<li>Wash twice in PBS for 5 min.</li>
	<li>Mount slides in mounting media containing DAPI.</li>
	<li>Capture images using a fluorescence or confocal microscope.</li>
</ol>

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The authors have nothing to disclose.

In this article we presented a method to assay and quantify the contribution of adipose tissue progenitor cells to the developing LNs and two techniques that allow the analysis of their progeny. Dissection of embryonic fat pads and newborns LNs are delicate and require manual skills gained by a lot of practice prior to the generation of the actual LN-fat pad chimera. To control the quality of the dissections, flow-cytometric analysis can be performed on the fat pads and LNs. Embryonic fat pad preparation should be...

Lymph nodes (LNs) are key organs of the immune system situated at strategic sites in the body, along the lymphatic vasculature network. They enable filtration of antigens and pathogens and provide a site for antigen presentation to lymphocytes and induction of adaptive immune responses. The stroma, which forms the basic structure of the LN and orchestrates the movement of the different hematopoietic participants of the adaptive immune response, is central to the function of these organs. Different populations of stromal cells supply essential and specific cues for the movements, localization, survival, proliferation and maturation of the hematopoietic component of the immune system1-3. Adult LN stromal cells fall in three categories: the blood endothelial cells, the lymphatic endothelial cells and fibroblasts. These three categories encompass heterogeneous populations. The fibroblastic populations contain fibroblastic reticular cells (FRC), follicular dendritic cells (FDC), marginal reticular cells (MRC), while the fibroblasts forming the capsule, the medulla and other cells which are not yet identified1-5. The origins and the mechanisms governing the maturation of the different LN stromal cell populations are unclear and the absence of specific markers allowing the fate mapping of specific LN stromal cell populations rends their study particularly difficult. However, a full understanding of the ontogeny of LN stromal cells is necessary to the comprehension of adaptive immune responses, the mechanisms contributing to tolerance and is at the basis of the development of artificial LNs.
So far, the study of LN stromal cell origin and development has been mostly limited to the direct assessment of LN development in embryos and newborns in wild type mice and different knockout mouse strains6-9. These approaches are limited by the embryonic and perinatal lethality of some of the mouse strains carrying deletions in genes important for LN development. Moreover, some of the genes essential for lymphoid tissue development are also involved in a wide range of biological process as it is the case for RANK10-11 or NF-&#954;B212. To address these issues, isolation and transplantation of embryonic LNs under the kidney capsule of a host mouse have been performed12-13. This technique allows, for example, the transfer of genetically modified embryonic LNs in a wild type environment to assess organ development and the recruitment and organization of host cells. However, the growth of embryonic LN grafted under the kidney capsule of an adult host is impaired, thus limiting the use of this technique.
LNs and fat deposits are anatomically closely associated and they develop simultaneously during embryogenesis. We recently demonstrated that the association LN/adipose tissue plays a crucial role in the provision of stromal cell progenitors for the LNs. In particular, signaling through the LT&#946;R controls the fate of adipocyte precursor cells by blocking adipogenesis and instead promoting maturation towards a LN stromal cell phenotype14. Here we describe a method based on generation of LN-fat pad chimeras allowing the tracing of adipose tissue derived cells in the developing LN. This method will be useful to determine the contribution of adipose tissues to different LN stromal cell populations and combined with the use of tissues from genetically modified mouse strains, will allow a better understanding of the mechanisms controlling the differentiation of the different LN stromal cell subsets.

<table><tbody><tr><td>2-Mercaptoethanol</td><td>Sigma</td><td>M3148</td><td></td></tr><tr><td>50 mm Sterilin Petri Dish</td><td>Appleton Woods</td><td>SC265</td><td></td></tr><tr><td>90 mm Sterilin Petri Dish</td><td>Appleton Woods</td><td>SC260</td><td></td></tr><tr><td>Adhesive slides</td><td>Surgipath</td><td>00202</td><td></td></tr><tr><td>Anti-mouse CD31 eFluor 450</td><td>eBioscience</td><td>48-0311</td><td></td></tr><tr><td>Anti-mouse CD4 Alexa 647</td><td>eBioscience</td><td>51-0041</td><td></td></tr><tr><td>Anti-mouse CD45 PerCP-Cy5.5</td><td>eBioscience</td><td>45-0451</td><td></td></tr><tr><td>Anti-mouse IgM Rhodamine Red</td><td>Stratech</td><td>715-296-020-JIR</td><td></td></tr><tr><td>Anti-mouse Podoplanin PE/Cy7</td><td>Biolegend</td><td>127411</td><td></td></tr><tr><td>Anti-mouse Podoplanin purified</td><td>eBioscience</td><td>14-5381</td><td></td></tr><tr><td>Collagenase D</td><td>Roche</td><td>11088858001</td><td></td></tr><tr><td>DMEM Medium</td><td>Sigma</td><td>D5671</td><td></td></tr><tr><td>DNase I</td><td>Sigma</td><td>DN25-1G</td><td></td></tr><tr><td>Dumont #5 Forceps Inox Biologie</td><td>FST</td><td>11252-20</td><td>Extra fine forceps for embryonic and newborn dissections</td></tr><tr><td>EDTA solution 0.5 M</td><td>Sigma</td><td>E7889</td><td></td></tr><tr><td>ERTR-7</td><td>Biogenesis</td><td></td><td></td></tr><tr><td>Fetal bovine serum</td><td>Sigma</td><td>F9665</td><td></td></tr><tr><td>Hardened fine iris scissors straight 11 cm</td><td>FST</td><td>14090-11</td><td>Small scissors for dissection</td></tr><tr><td>HEPES solution</td><td>Sigma</td><td>H0887</td><td></td></tr><tr><td>Isopore Membrane Filters 0.8 &amp;mu;m ATTP</td><td>Millipore</td><td>ATTPO 1300</td><td></td></tr><tr><td>L-Glutamine solution</td><td>Sigma</td><td>G7513</td><td></td></tr><tr><td>MEM Nonessential Amino Acid Solution (100x)</td><td>Sigma</td><td>M7145</td><td></td></tr><tr><td>O.C.T. Compound</td><td>Tissue-Tek</td><td>4583</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma</td><td>P4458</td><td></td></tr><tr><td>Plastic box with lid</td><td>Watkins and Doncaster</td><td>E6052</td><td>Sandwich box for &lt;em&gt;in vitro&lt;/em&gt; organ culture. Drill two holes in the lid to allow gas exchange in the CO&lt;sub&gt;2&lt;/sub&gt; incubator.</td></tr><tr><td>RPMI 1640 Medium</td><td>Sigma</td><td>R0883</td><td></td></tr><tr><td>Sponges Vulkan Underwrap</td><td>Patterson Medical</td><td>004383</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>LEICA MZ95</td><td>Dissecting microscope with zoom</td></tr><tr><td>Thermomixer</td><td>Eppendorf</td><td>5436</td><td></td></tr><tr><td>Vectashield Mounting Medium with DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr></tbody></table>

generation of lymph node-fat pad chimeras for the study of lymph node stromal cell origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Three weeks after transplantation, the LN/fat pad chimera is recovered from the kidney. The chimera is now very similar to a normal LN in its own fat pad, and the LN is visible in the center of the adipose tissue (Figure 1). If the LN can&#39;t be identified, it is possible that it was lost during the transplantation on the kidney. When assessing the role of genes potentially important in LN development, the LNs recovered may remain very small and more difficult to find. This is the case when adipose tis...

Watch this Scientific Journal Video about Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin at JoVE.com

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

generation-lymph-node-fat-pad-chimeras-for-study-lymph-node-stromal

Research

JoVE Journal

Immunology and Infection

6.0K Views.  University of Birmingham. Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

Video: Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the initiation of an immune response 1. Here, we present methods for adoptive transfer of labeled T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node as first described in 2002 2. Two-photon imaging of immune cells requires that the cells are fluorescently labeled, either by staining with a cell tracker dye or by expressing a fluorescent protein. We demonstrate the adoptive transfer procedure of injecting cells derived from donor mice into the tail vein of a recipient animal, where they home to lymphoid organs within approximately 15-30 min. We illustrate the isolation of a lymph node and describe methods to ensure proper mounting of the excised lymph node. Other considerations such as proper oxygenation of perfused media, temperature, and laser power are discussed. Finally, we present 3D video images of naive CD4+ T cells exhibiting steady state motility at 37&deg;C.

Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1. Here, we demonstrate adoptive transfer of T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node.

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the ...

Biology

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Cancer Research

Reaggregate Thymus Cultures

Stromal cells within lymphoid tissues are organized into three-dimensional structures that provide a scaffold that is thought to control the migration and development of haemopoeitic cells. Importantly, the maintenance of this three-dimensional organization appears to be critical for normal stromal cell function, with two-dimensional monolayer cultures often being shown to be capable of supporting only individual fragments of lymphoid tissue function. In the thymus, complex networks of cortical and medullary epithelial cells act as a framework that controls the recruitment, proliferation, differentiation and survival of lymphoid progenitors as they undergo the multi-stage process of intrathymic T-cell development. Understanding the functional role of individual stromal compartments in the thymus is essential in determining how the thymus imposes self/non-self discrimination. Here we describe a technique in which we exploit the plasticity of fetal tissues to re-associate into intact three-dimensional structures in vitro, following their enzymatic disaggregation. The dissociation of fetal thymus lobes into heterogeneous cellular mixtures, followed by their separation into individual cellular components, is then combined with the in vitro re-association of these desired cell types into three-dimensional reaggregate structures at defined ratios, thereby providing an opportunity to investigate particular aspects of T-cell development under defined cellular conditions. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

In this video the preparation of 2-dGuo-treated reaggregate thymus cultures is demonstrated.

Stromal cells within lymphoid tissues are organized into three-dimensional structures that provide a scaffold that is thought to control the migration and ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during inflammatory responses. Unfortunately, study of pDC function is hindered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation predominantly favor the production of cDCs over pDCs. Here we present a unique approach to efficiently generate pDCs from common lymphoid progenitors (CLPs) in vitro. Specifically, the protocol described details how to purify CLPs from bone marrow and generate pDCs by coculturing with &#947;-irradiated AC-6 feeder cells in the presence of Flt3 ligand. A unique characteristic of this culture system is that the CLPs migrate underneath the AC-6 cells and become cobblestone area-forming cells, a critical step for expanding pDCs. Morphologically distinct DCs, namely pDCs and cDCs, were generated after approximately 2 weeks with a composition of 70-90% pDCs under optimal conditions. Typically, the number of pDCs generated by this method is roughly 100-fold of the number of CLPs seeded. Therefore, this is a novel system with which to robustly generate the large numbers of pDCs required to facilitate further studies into the development and function of these cells.

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Medicine

Basement Membrane Matrix Encapsulated Cell Aggregation for Investigating Murine Spleen Tissue Formation

The spleen is an immune organ that plays a key role in blood-borne immune responses. The anatomical or functional loss of this tissue increases susceptibility to severe blood infections and sepsis. Auto-transplantation of spleen slices has been used clinically to replace lost tissue and restore immune function. However, the mechanism driving robust and immunologically functional spleen tissue regeneration has not been fully elucidated. Here, we aim to develop a method for aggregating and encapsulating spleen cells within a semi-solid matrix in order to investigate the cellular requirements for spleen tissue formation. Basement membrane matrix encapsulated cell constructs are amenable to both in vitro tissue culture of three-dimensional organoids as well as transplantation under the kidney capsule to directly assess in vivo tissue formation. By manipulating the input cells for aggregation and encapsulation, we demonstrate that graft-derived PDGFR&#946;+MAdCAM-1- neonatal stromal cells are required for spleen tissue regeneration under animal transplantation models.

This article describes a protocol for aggregating and encapsulating spleen cells within a semi-solid basement membrane matrix. Basement membrane matrix constructs can be used in three-dimensional culture for studying organoid development, or for in vivo transplantation and tissue regeneration studies.

The spleen is an immune organ that plays a key role in blood-borne immune responses. The anatomical or functional loss of this tissue increases ...

Developmental Biology

Interrogating Individual Autoreactive Germinal Centers by Photoactivation in a Mixed Chimeric Model of Autoimmunity

Autoimmune diseases present a significant health burden. Fundamental questions regarding the development and progression of autoimmune disease remain unanswered. One requirement for advancements in our understanding of the underlying disease mechanisms and cellular dynamics is the precise coupling of the microanatomical location of cell subsets with downstream molecular or functional analyses; a goal that has traditionally been difficult to achieve. The development of stable photoactivatable biological fluorophores and their integration into reporter strains has recently enabled precise microanatomical labeling and tracking of cellular subsets in murine models. Here, we describe how the ability to analyze autoreactive lymphocytes from single germinal centers may help to provide novel insights into autoimmunity, using the combination of a novel chimeric model of autoimmunity with a photoactivatable reporter as an example. We demonstrate a procedure for generating mixed chimeras with spontaneous autoreactive germinal centers populated by lymphocytes carrying a photoactivatable green fluorescent protein reporter. Using in vivo labeling strategies, single germinal centers can be visualized in explanted lymphoid tissues and their cellular constituents photoactivated by two-photon microscopy. Photoactivated lymphocytes from single germinal centers can then be analyzed or sorted flow cytometrically, as single cells or in bulk, and may be subjected to additional downstream molecular and functional analyses. This approach may directly be applied to provide renewed insights in the field of autoimmunity, but the procedure for generating bone marrow chimeras and the photoactivation procedure may additionally find broad application in studies of infectious diseases and tumor metastases.

This protocol describes the generation of mixed murine bone marrow chimeras with spontaneous autoimmune germinal centers, in which autoreactive lymphocytes carry a photoactivatable green fluorescent protein (PA-GFP) reporter. This provides the ability to link cellular location in tissues with downstream molecular and functional analyses.

Autoimmune diseases present a significant health burden. Fundamental questions regarding the development and progression of autoimmune disease remain ...

Evaluating Antigen-Specific CD8+ T Cells During Lymph Node Metastasis

Draining Lymph Node Metastasis Model for Assessing the Dynamics of Antigen-Specific CD8+ T Cells During Tumorigenesis

Tumor antigen-specific CD8+ T cells from draining lymph nodes gain an accumulating importance in mounting anti-tumor immune response during tumorigenesis. However, in many cases, cancer cells form metastatic loci in lymph nodes before further metastasizing to distant organs. To what extent the local and systematic CD8+ T cell responses were influenced by LN metastasis remains obscure. To this end, we set up a murine LN metastasis model combined with a B16F10-GP melanoma cell line expressing the surrogate neoantigen derived from lymphocytic choriomeningitis virus (LCMV), glycoprotein (GP), and P14 transgenic mice harboring T cell receptors (TCRs) specific to GP-derived peptide GP33-41 presented by the class I major histocompatibility complex (MHC) molecule H-2Db. This protocol enables the study of antigen-specific CD8+ T cell responses during LN metastasis. In this protocol, C57BL/6J mice were subcutaneously implanted with B16F10-GP cells, followed by adoptive transfer with naive P14 cells. When the subcutaneous tumor grew to approximately 5 mm in diameter, the primary tumor was excised, and B16F10-GP cells were directly injected into the tumor draining lymph node (TdLN). Then, the dynamics of CD8+ T cells were monitored during the process of LN metastasis. Collectively, this model has provided an approach to precisely investigate the antigen-specific CD8+ T cell immune responses during LN metastasis.

Author Spotlight: A Model to Study the Systemic and Local Dynamics of CD8+ T Cells During LN Metastasis

The experimental design presented here provides a useful reproductive model for the studies of antigen-specific CD8+ T cells during lymph node (LN) metastasis, which excludes the perturbation of bystander CD8+ T cells.

Tumor antigen-specific CD8+ T cells from draining lymph nodes gain an accumulating importance in mounting anti-tumor immune response during tumorigenesis. ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

Summary

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Chapters in this video

Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

Reaggregate Thymus Cultures

Isolation of Murine Lymph Node Stromal Cells

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Basement Membrane Matrix Encapsulated Cell Aggregation for Investigating Murine Spleen Tissue Formation

Interrogating Individual Autoreactive Germinal Centers by Photoactivation in a Mixed Chimeric Model of Autoimmunity

Draining Lymph Node Metastasis Model for Assessing the Dynamics of Antigen-Specific CD8⁺ T Cells During Tumorigenesis