The authors are grateful for the experimental help in developing the method of intralymphatic immunization in mice from Iris Erdmann, Barbara von Beust, and Julia Maria Mart&#237;nez-G&#243;mez. Thanks also to Maggy Arras and Nikola Cesarovic for letting us use their surgical theatre for this video production.&#160;

1. Anesthesia of Mice
<ol>
	<li>Prepare the anesthetics by mixing ketamine (dissociative anesthetic) and xylazine (sedative and analgesic) in buffered saline. The concentrations of ketamine and xylazine in the final solution are 12.5 and 2 mg/ml, respectively.</li>
	<li>Inject the anesthetics to the mice by intraperitoneal administration using a syringe with a 25-30 G needle. Use 0.1 ml/10 g of body weight.</li>
	<li>Apply an ophthalmic ointment to the mice&#8217;s eyes in order to prevent drying-out of the corneas.</li>
	<li>Assure that the mouse is sufficiently anesthetized by pinching its foot or toe with the forceps. If the mouse does not react with reflexes to the pinch, proceed to surgery. If the mouse reacts to the pinch with reflexes or muscle contractions, wait 1-2 min and repeat the pinch test. If the mouse still reacts, replace the mouse with a new animal and repeat the procedures from step 1.2 onwards.</li>
</ol>
2. Surgical Incision of the Inguinal Area of the Mouse
<ol>
	<li>Place the mouse on its back.</li>
	<li>Wet one inguinal region with disinfecting ethanol (70%). To provide better disinfection, the inguinal region may be shaved to remove fur prior disinfection with ethanol.</li>
	<li>Take the hind leg and bend the hip joint to produce an approximately 90&#176; angle of the hip joint.</li>
	<li>Using a curved microdissecting forceps, take the mouse skin in the inguinal area where the hip joint is bent and pull the wetted skin up slightly.</li>
	<li>While holding the skin up with the forceps, cut a small incision (&#60;5 mm) through the skin using surgical scissors.</li>
	<li>Place the tip of the closed scissors into the incision and further open the incision by opening the scissors while holding it inside the incision. This will cause the skin to tear and should produce a diameter of less than 10 mm. Note: A cold light source with flexible light guides can be used to improve visibility. In older mice, especially males, the identification of the lymph node may be difficult due to much fat tissue in the inguinal region.</li>
</ol>
3. Intralymphatic Injection
<ol>
	<li>Prepare a syringe (0.5 ml or less) with a 28-30 G hypodermic needle; a short bevel may be preferable to a long bevel. Aspirate 10 &#956;l of the vaccine to be injected and assure that the syringe is free of air.</li>
	<li>Localize the inguinal lymph node with help of the curved forceps and the tip of the closed scissor. The lymph node will appear greyish within the more whitish fat tissue and can be further identified through afferent and efferent capillaries entering and leaving the lymph node.</li>
	<li>Immobilize the lymph node by holding it between the branches of the curved forceps.</li>
	<li>Take the syringe and insert the needle into the lymph node with the bevel facing up; making sure the whole bevel enters the lymph node.</li>
	<li>Inject the vaccine (10 &#956;l). If the lymph node is swelling (blowing up), the injection can be assumed successful. If the needle is not placed deep enough into the lymph node, some or the entire vaccine may leak out and not enter the lymph node. If the needle is inserted too deep, the injected vaccine will be released subcutaneously below the lymph node. In case of the latter, repeat the injection or replace the mouse with a new untreated mouse.</li>
</ol>
4. Closing the Incision by Suturing
<ol>
	<li>Open a pack of sterile surgical suture.</li>
	<li>Take hold of the suture needle with a needle holder at the distal portion of the needle body. Tighten the needle holder by squeezing it until the first ratchet catches.</li>
	<li>Take hold of the skin at one side of the incision using forceps (depending on the surgeon's preference, toothed or un-toothed forceps or skin hooks may be used). Insert the needle ca. 2-3 mm from the edge of the incision, entering from the outside of the skin.</li>
	<li>Loosen hold of the skin with the forceps and grasp the opposite side of the incision with the forceps. Insert the needle ca. 2-3 mm from the edge of the incision, entering from the inside of the skin.</li>
	<li>Loosen the needle holder and grasp the needle end with the forceps. Pull the needle with the tread through the skin, leaving a convenient length of the tread outside the first needle-insertion point (if several mice are to be treated, leave a shorter end; 5-10 mice can be operated on with one suture needle/tread).</li>
	<li>Make the preferred knot according to standard surgeon protocols. One stitch is sufficient if the incision is not larger than 10 mm. For larger incisions, make a second stitch.</li>
</ol>
5. Post-operational Treatment
<ol>
	<li>Place the mouse in the cage and cover with tissues to keep warm. When treating multiple mice, place them close to each other to keep warm. Alternatively, place the mice on a warm pad until they have woken up.</li>
	<li>Observe the mice until they wake up.</li>
	<li>Observe the mice daily with regards to wound healing as well as other clinical symptoms such as wound infections. The wound is typically sufficiently closed within two days and healed within seven days of the procedure.</li>
</ol>

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TMK is named as inventor of patents dealing with intralymphatic immunotherapy in man. TMK has been scientific advisor and has received travel expenses from ImVisioN GmbH, Cytos Biotechnology, MannKind Corporation,&#160;and XBiotech USA Inc. PJ has no conflict interest to disclose.

Intralymphatic immunization and immunotherapy have been shown to be appropriate for stimulation of both antibody responses and T-cell responses. As demonstrated in this video-article, the intralymphatic procedure of vaccination is a quick and easy method for stimulating strong immune responses in mice. A trained surgeon can perform the procedure during 3-4 min. The session can also be shared between two surgeons where one is typically doing anesthesia and the suturing and the second surgeon is doing the incision and the ...

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this procedure requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of T- and B-cell responses. In addition, the strength and the type of immune responses induced also depend on the density or frequency of such interactions as well as the microenvironment itself, especially the content of cytokines. As only a small fraction of a vaccine injected into a peripheral tissue reaches a lymph node, vaccinations of mice and humans by direct injection of the vaccine into lymph nodes, the site were immune responses are generated, have been performed. In man, the procedure is guided by ultrasound, a procedure also used for the administration of imaging agents for visualization and diagnosis in the lymphatic system. In mice, the procedure is invasive. Here, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals1, the lymph node is localized and immobilized with forceps, and a volume of 10-20 &#956;l of the vaccine is injected under visual control; 10 &#956;l is used for first injections and in young mice with small lymph nodes, whereas 20 &#956;l can be injected into lymph nodes of older or already primed mice, which have larger lymph nodes. The incision can be closed with a single stitch using surgical sutures. By this method, mice have vaccinated with plasmid DNA2,3, messenger RNA4, peptide1,3,5,6, protein7-10, particles11, bacteria12 as well as adjuvants7,13, and strong improvement of immune responses against all type of vaccines have been observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited or very costly. In human, the intralymphatic method of immunization has been applied to allergy patients14,15 or to patients with cancer16-21. Although the current notion is that the intralymphatic method is more invasive than other injectable methods such as intramuscular and subcutaneous injections, the pain perception is not higher than after a venous puncture15. It is expected that intralymphatic vaccination will become an alternative or complement to other methods of prophylactic and especially therapeutic vaccinations. This article describes in details how the procedure of intralymphatic vaccination is performed in mice. All procedures described, were approved by the Cantonal Veterinary Agency of Zurich and performed according Swiss federal guidelines and directives on the protection of animals used for scientific purposes.

<table><tbody><tr><td>Ketamine (Ketasol-100)</td><td>Graeub AG, Switzerland</td><td></td><td>Anesthetics</td></tr><tr><td>Xylazine (Rompun)</td><td>Bayer, Germany</td><td></td><td>Anesthetics</td></tr><tr><td>Viscotears Eye-Gel</td><td>Novartis, Switzerland</td><td></td><td>To keep eyes from drying out during anesthesia.</td></tr><tr><td>BD Micro-Fine 0.5 ml</td><td>BD Medical, France</td><td></td><td>29 G Insulin syringes with permanently attached needles</td></tr><tr><td>6-0 Dermalon Monofilament Nylon</td><td>Covidien, MA, USA</td><td></td><td>For sutures (0.7 metric, 18 G, 45 cm, Blue)</td></tr><tr><td>Curved forceps, 4.5 in</td><td>Polymed, Switzerland</td><td></td><td>For incision and holding of lymph node</td></tr><tr><td>Straight surgical scissors, 4.5 in</td><td>Polymed, Switzerland</td><td></td><td>For incision</td></tr><tr><td>Needle holder, 5.5 in</td><td>Polymed, Switzerland</td><td></td><td>To close incision with suture</td></tr></tbody></table>

intralymphatic immunotherapy and vaccination in mice

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e.&#160;intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 &#956;l of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.

The procedure of intralymphatic injections in mice, despite the surgical nature, is straight forward and relatively fast. A trained person can perform the procedure in 3-4 min. The incision that is closed with one stitch typically heals within two days (Figure 1)
Intralymphatic vaccination or immunization has been performed with mRNA, plasmid DNA, peptides, proteins, virus and bacteria. Figure 2 illustrates the antibody production after immunization with the p...

Watch this Scientific Journal Video about Intralymphatic Immunotherapy and Vaccination in Mice at JoVE.com

Intralymphatic Immunotherapy and Vaccination in Mice

Prophylactic and therapeutic vaccination often fails to stimulate strong immune responses due to week drainage of the vaccine to lymph nodes and consequently poor involvement of immune cells. By direct injection of vaccine to lymph nodes, so-called intralymphatic injection, vaccine efficacy can be strongly improved and vaccine doses can be reduced.

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of ...

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Immunology and Infection

23.0K Views.  University Hospital Zurich. Prophylactic and therapeutic vaccination often fails to stimulate strong immune responses due to week drainage of the vaccine to lymph nodes and consequently poor involvement of immune cells. By direct injection of vaccine to lymph nodes, so-called intralymphatic injection, vaccine efficacy can be strongly improved and vaccine doses can be reduced.

Video: Intralymphatic Immunotherapy and Vaccination in Mice

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an 
appropriate immune response to a variety of immunogens1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be 
administered intranasally1,3,4, intratracheally5 or as aerosols6. Delivery by each route involves distinct technical skills and limitations that need to be 
considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory 
tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)7, potentially complicating interpretation of the results. In addition, swallowing, 
sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may 
be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) 
route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the 
trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t 
instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the 
innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

Intra-lymph Node Injection of Biodegradable Polymer Particles

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g.&#160;size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.

Lymph nodes are the immunological tissues that orchestrate immune response and are a critical target for vaccines. Biomaterials have been employed to better target lymph nodes and to control delivery of antigens or adjuvants. This paper describes a technique combining these ideas to inject biocompatible polymer particles into lymph nodes.

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these ...

Bioengineering

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Medicine

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been used in tumor vaccines to activate innate immunity and enhance antitumor responses. This protocol demonstrates a therapeutic model using cell-based tumor vaccine consisting of Flt3L-expressing B16-F10 melanoma cells along with phenotypic and functional analysis of immune cells in the tumor microenvironment (TME). Procedures for cultured tumor cell preparation, tumor implantation, cell irradiation, tumor size measurement, intratumoral immune cell isolation, and flow cytometry analysis are described. The overall goal of this protocol is to provide a preclinical solid tumor immunotherapy model, and a research platform to study the relationship between tumor cells and infiltrating immune cells. The immunotherapy protocol described here can be combined with other therapeutic modalities, such as immune checkpoint blockade (anti-CTLA-4, anti-PD-1, anti-PD-L1 antibodies) or chemotherapy in order to improve the cancer therapeutic effect of melanoma.

The protocol presents a cancer immunotherapy model using cell-based tumor vaccination with Flt3L-expressing B16-F10 melanoma. This protocol demonstrates the procedures, including preparation of cultured tumor cells, tumor implantation, cell irradiation, measurement of tumor growth, isolation of intratumoral immune cells, and flow cytometry analysis.

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been ...

Cancer Research

Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung&#8217;s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages. 
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs&#8217; immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.

The present work illustrates the convenience of using sublingual immunotherapy to boost the innate immune response in the lungs and confer protection against acute pneumococcal pneumonia in mouse.

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data have encouraged pulmonary tuberculosis vaccines as a promising strategy to prevent pulmonary disease, which is responsible for transmission. In this work, we describe the methodology used to demonstrate in the mouse model the benefits of intranasal BCG vaccination when compared to subcutaneous. Our data revealed greater protective efficacy following intranasal BCG administration. In addition, our results indicate that pulmonary vaccination triggers a higher immune response in lungs, including Th1 and Th17 responses, as well as an increase of immunoglobulin A (IgA) concentration in respiratory airways. Our data show correlation between protective efficacy and the presence of IL17-producing cells in lungs post-Mycobacterium tuberculosis challenge, suggesting a role for this cytokine in the protective response conferred by pulmonary vaccination. Finally, we detail the global workflow we have developed to study respiratory vaccination in the mouse model, which could be extrapolated to other tuberculosis vaccines, apart from BCG, targeting the mucosal response or other pulmonary routes of administration such as the intratracheal or aerosol.

We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

Antigenic Liposomes for Generation of Disease-specific Antibodies

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for vaccination as well as understand how pathogenic antibody responses develop in allergies and autoimmune disease. Generating robust antigen-specific antibody responses is not always trivial. In mouse models, it often requires multiple rounds of immunizations with adjuvant that leads to a great deal of variability in the levels of induced antibodies. One example is in mouse models of peanut allergies where more robust and reproducible models that minimize mouse numbers and the use of adjuvant would be beneficial. Presented here is a highly reproducible mouse model of peanut allergy anaphylaxis. This new model relies on two key factors: (1) antigen-specific splenocytes are adoptively transferred from a peanut-sensitized mouse into a na&#239;ve recipient mouse, normalizing the number of antigen-specific memory B- and T-cells across a large number of mice; and (2) recipient mice are subsequently boosted with a strong multivalent immunogen in the form of liposomal nanoparticles displaying the major peanut allergen (Ara h 2). The major advantage of this model is its reproducibility, which ultimately lowers the number of animals used in each study, while minimizing the number of animals receiving multiple injections of adjuvant. The modular assembly of these immunogenic liposomes provides relatively facile adaptability to other allergic or autoimmune models that involve pathogenic antibodies.

Described is the preparation of antigenic liposomal nanoparticles and their use in stimulating B-cell activation in vitro and in vivo. Consistent and robust antibody responses led to the development of a new peanut allergy model. The protocol for generating antigenic liposomes can be extended to different antigens and immunization models.

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for ...

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 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 ...

A Minimally Invasive, Accurate, and Efficient Technique for Intrathymic Injection in Mice

Intrathymic injection in mouse models is an important technique for studying thymic and immune function, including genetic and acquired T cell disorders. This requires methods for the direct deposition of reagents and/or cells into the thymus of living mice. Traditional methods of intrathymic injection include thoracic surgery or minimally invasive percutaneous blind injections, both of which have significant limitations. Ultra-high frequency ultrasound imaging devices have made image-guided percutaneous injections possible in mice, greatly improving the injection accuracy of the percutaneous injection approach and enabling the injection of smaller targets. However, image-guided injections rely on the utilization of an integrated rail system, making this a rigid and time-consuming procedure. A unique, safe, and efficient method for percutaneous intrathymic injections in mice is presented here, eliminating reliance on the rail system for injections. The technique relies on using a high-resolution micro-ultrasound unit to image the mouse thymus noninvasively. Using a free-hand technique, a radiologist can place a needle tip directly into the mouse thymus under sonographic guidance. Mice are cleaned and anesthetized before imaging. For an experienced radiologist adept at ultrasound-guided procedures, the learning period for the stated technique is quite short, typically within one session. The method has a low morbidity and mortality rate for the mice and is much faster than current mechanically assisted techniques for percutaneous injection. It allows the investigator to efficiently perform precise and reliable percutaneous injections of thymuses of any size (including very small organs such as the thymus of aged or immunodeficient mice) with minimal stress on the animal. This method enables the injection of individual lobes if desired and facilitates large-scale experiments due to the time-saving nature of the procedure.

The present protocol describes an interventional radiology procedure established for intrathymic injection in mice to avoid the risk of open surgery and improve the accuracy of blind percutaneous injections.

Intrathymic injection in mouse models is an important technique for studying thymic and immune function, including genetic and acquired T cell disorders. ...

Tumor Implantation and Injection of a Tumor Vaccine in a Mouse Model

Source: Liu, H. Y., et al. <a href="https://app.jove.com/v/64082">Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine.</a> J. Vis. Exp. (2023).
This video demonstrates a cell-based tumor vaccination assay. A mouse, intradermally injected with B16-F10 melanoma cells for tumor formation, is vaccinated with B16-F10 cells secreting Flt3L cytokine near the developed tumor. The secreted Flt3L from the vaccine promotes the maturation and proliferation of dendritic cells that then present tumor antigens to T cells in lymph nodes, triggering a targeted anti-tumor immune response.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
All mice ...