We would like to thank Mads Radmer Jensen for his practical and academic advice; Lau Lindqvist and Kaare Svejstrup for providing technical expertise; Helene B&#230;k Juel, Jonathan Filskov, Signe T. Schmidt, and Solveig W. Harp&#248;th for their technical assistance; the staff at the Gentofte TB outpatient clinic for their training in PPD administration; and all the volunteers for participating in the study. This project is funded by the European Commission H2020 program [grant number TBVAC2020 643381], and we would like to thank consortium partners for the fruitful discussions.

All methods described below, including the use of human volunteers, have been approved by the Danish Committee on Health Research Ethics (H-15002988) and the Danish Data Protection Agency (jr.nr. 2015-57-0102). PPD must be a certified product approved for human use and administered within the correct dosage provided by the manufacturer. Any deviation in dosage or administration may require additional ethical approvals and volunteers must give informed consent.
1. Tuberculin Skin Test
<ol>
	<li>Obtain oral and written informed consent from the volunteer. Prior to the injection, ensure that the volunteer understands and accepts the procedure including the possible adverse effects. 
	NOTE: Inclusion criteria specific to this protocol are age &#62; 18 years and a documented BCG vaccination. Inclusion criteria may vary depending on the research question (i.e., an additional inclusion criterion could be evidence of positive TST).</li>
	<li>Use a ready-made solution of PPD for human use [20 tuberculin units (TU)/mL]. Disinfect the rubber cap of a vial using an alcohol swab.</li>
	<li>Prepare the injection site
	<ol>
		<li>Locate the injection site on the ventral side of the volunteer&#39;s forearm.</li>
		<li>Place the arm palm-up on a plain surface and choose an area on the upper third of the forearm, approximately 5 - 10 cm from the elbow joint. Stay clear of scars, veins, or damaged skin.</li>
		<li>Disinfect the area using an alcoholic swab.</li>
	</ol>
	</li>
	<li>Prepare the PPD-solution
	<ol>
		<li>Use a 1 mL sterile syringe with a 27 - 30G/short (e.g., 12 mm) fixed needle.</li>
		<li>Gently mix and aspire 0.1 mL of the PPD solution. Make sure that there are no visible bubbles.</li>
	</ol>
	</li>
	<li>Intradermal PPD injection 
	NOTE: This step describes how to perform a single PPD deposition, but multiple depositions of PPD in the same arm is possible. However, this may require specific ethical approval.
	<ol>
		<li>Stretch the skin and point the needle at a 5 - 15&#176; angle to the skin with the bevel facing upwards.</li>
		<li>Insert the tip of the needle into the dermal layer of the skin and make sure the tip is almost visible through the epidermis.</li>
		<li>Slowly inject 0.1 mL of PPD solution. 
		NOTE: If administered correctly, a pale papule of 6 - 10 mm will appear immediately. The papule disappears after approximately 10 min.</li>
		<li>When making two or more depositions, aim for a maximum separation (5 - 10 cm) of the injection sites while keeping in a good distance from the elbow and wrist area. 
		NOTE: This prevents a potential confluence of the skin test response and allows the uninterrupted positioning of two suction chambers.</li>
	</ol>
	</li>
</ol>
2. Evaluation of the Skin Reaction - Day 3
<ol>
	<li>After 48 - 72 h, note the size of the skin reaction. Measure the induration (the swelling) and not the erythema (the redness) of the reaction.</li>
	<li>Palpate the hard swelling using fingers and then mark the edges of the induration using a ballpoint pen. Use a ruler to measure the induration diameter and note the result in mm. 
	NOTE: A reading of the induration size may guide the choice of orifice diameter for the suction chamber.</li>
</ol>
3. Suction Blister Induction - Day 7
<ol>
	<li>Assemble the suction device unit. 
	Note: Suction device units are vacuum pumps with attached chambers for suction blister induction. The device used in this demonstration is custom made, but suction device units are also commercially available. The pump needs to be adjustable within the range of -20 to -40 kPa and provide a steady and reliable negative pressure. Regional ethical approval should be obtained prior to conducting experiments using this method.
	<ol>
		<li>First, prepare the suction chamber by assembling the bottom orifice plate and the window plate with the chamber and the connecting hose. Attach the chamber-connecting hose tightly to the vacuum pump. 
		Note: The chambers should include a bottom plate with a hole for the blister induction, which should be suitable for contact with the human skin, and a top plate with a window, allowing for the visual monitoring of the blister.</li>
	</ol>
	</li>
	<li>Determine the optimal orifice diameter of the orifice plate relative to the size of the skin reaction; the larger the size of the orifice, the larger the blister.</li>
	<li>Disinfect the orifice plate and the skin of the volunteer using alcohol swabs.</li>
	<li>Attach the suction chamber to the skin.
	<ol>
		<li>Make sure that the volunteer&#39;s arm is resting comfortably.</li>
		<li>Make sure that the hole in the bottom plate of the suction chamber is situated over the PPD reaction so that the center of the skin reaction is visible.</li>
		<li>Secure the chamber loosely using straps: when negative pressure is applied, the chamber will adhere to the skin and maintain its position.</li>
	</ol>
	</li>
	<li>Turn the suction device on and adjust the pressure to -20 kPa.</li>
	<li>After 30 min, increase the negative pressure to -25 kPa.</li>
	<li>After 60 min, further increase the negative pressure to -30 kPa.</li>
	<li>Keep the pressure at -30 kPa until a blister is fully formed. Ensure that the negative pressures provided in this protocol are specific to a customized instrument. It may be necessary to adjust the pressure slightly when applying the protocol in other settings. 
	NOTE: The blister induction phase ranges from 60 to 180 min (1 - 3 h) with the actual blister formation occurring within the last 30 min. Blistering is often associated with an itchy sensation. The blisters may occur as single or multiple merging blisters. If blister rupture or hemorrhage occurs, it is advised to terminate the blister induction by slowly releasing the pressure.</li>
	<li>After the blister is fully formed, release the pressure, and carefully remove the suction chamber from the skin without rupturing the blister.</li>
	<li>Apply a soft dressing on the surrounding skin area and place a hard cap (e.g., from a 50 mL plastic tube) over the blister.</li>
	<li>Secure the cap with non-allergenic surgical tape followed by a soft stretchy bandage.</li>
	<li>Instruct the volunteer to avoid excessive physical strain on the blister area for the next 12 - 24 h.</li>
</ol>
4. Harvest of Blister Fluid - Day 8
<ol>
	<li>On the 8th day, gently remove the protective dressing and disinfect the blister with skin disinfection spray.</li>
	<li>Use a 2 mL sterile syringe with a 23G needle to harvest the blister content. Insert the needle into the top/lateral side of the blister roof and slowly aspirate the fluid. Avoid touching the floor of the cavity but make sure to harvest all the fluid.</li>
	<li>Apply a dressing on the collapsed blister.</li>
	<li>Transfer the blister fluid to a sterile tube. Note the volume.</li>
	<li>Spin the blister fluid down at 600 x g using a tabletop centrifuge for 4 min.</li>
	<li>Transfer the supernatant to another tube and resuspend the cell pellet in 500 &#181;L of cell medium. 
	NOTE: The cells are now ready for counting and further analysis. Supernatants can be stored at -20 to -80 &#176;C until use.</li>
</ol>
5. Analyses of Suction Blister Cells and Fluids
NOTE: Providing instructions for the analysis of cells and fluid obtained from skin suction blisters is not within the scope of this protocol. However, in order to provide representative results for this report, intracellular stain flow cytometry and multiplex analyses were performed. The methodology is described in brief below.
<ol>
	<li>Flow cytometry
	<ol>
		<li>Stimulate suspensions of 1 x 105 fresh cells isolated from suction blisters from 1 BCG-vaccinated volunteer with 10 &#181;g/mL of PPD and incubate the cells at 37 &#176;C and 5% CO2. Include unstimulated cells for a parallel incubation.</li>
		<li>Stimulate suspensions 1 x 106 PBMCs obtained from 1 BCG-vaccinated volunteer with 10 &#181;g/mL of PPD and incubate the mixture at 37 &#176;C and 5% CO2. Include unstimulated cells for a parallel incubation.</li>
		<li>After 2 h, treat all cells with Brefaldin A and monensin and incubate them for 6 h at 37&#176;C.</li>
		<li>Stain the cells with a panel of Live/Dead-stain-BV510, anti-CD4-AF780, anti-CD3-BV421, anti-CD8-PerCP-Cy5.5, anti-CCR7-PE, anti-CD45RA-BV786, anti-IFN-&#947;-AF700, anti-TNF-&#945;-PE-Cy7, and anti-IL-2-FITC.</li>
		<li>Include FMO controls in the PBMC panel.</li>
		<li>Perform a flow cytometric analysis with a flow cytometer and analyze the results using relevant software.</li>
		<li>Gate on the cytokine-producing CD3+CD4+CD8- subsets using the following gating strategy: singlets - &#62; viable CD3+ - &#62; CD4+CD8- - &#62; IFN-&#947;+, TNF-&#945;+, or IL-2+.</li>
		<li>Gate on effector memory cell populations using the following gating strategy: singlets - &#62; viable CD3+ - &#62; CD4+CD8- - &#62; CCR7-CD45RA-.</li>
		<li>Calculate individual cytokine profiles using Boolean gating.</li>
	</ol>
	</li>
	<li>Cytokine release demonstrations
	<ol>
		<li>For a demonstration of a cytokine release in vivo, use a multiplex analysis to quantify the levels of IL-2, IFN-&#947;, TNF-&#945;, IL-10, and IL-13 in thawed suction blister fluid obtained from 4 volunteers.</li>
		<li>For a demonstration of a cytokine release by cells obtained from suction blisters ex vivo, use fresh suction blister cells and PBMCs obtained from 1 BCG-vaccinated volunteer.</li>
		<li>Infect the PBMC and suction blister cells with 100 colony-forming units (CFU) of Mtb H37Rv and incubate them for 4 days.</li>
		<li>Use a multiplex analysis to quantify the levels of IL-2, IFN-&#947;, TNF-&#945;, IL-10, and IL-13 in all culture supernatants.</li>
		<li>Adjust the measurements above the assay calculation range to the upper calculation limit.</li>
	</ol>
	</li>
</ol>

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(110), e52564 (2016).</li><li>Koskela, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blister+fluid+and+serum+cytokine+levels+in+severe+sepsis+in+humans+reflect+skin+dysfunction.">Blister fluid and serum cytokine levels in severe sepsis in humans reflect skin dysfunction.</a> Acta Anaesthesiologica Scandinavica. 61 (1), 53-61 (2017).</li><li>Wada, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+skin+blister+fluids+from+children+with+Epstein-Barr+virus-associated+lymphoproliferative+disease.">Characterization of skin blister fluids from children with Epstein-Barr virus-associated lymphoproliferative disease.</a> The Journal of Dermatology. 45 (4), 444-449 (2018).</li><li>Andersson, C., Rajala, T., S&#228;rkk&#228;, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hierarchical+models+for+epidermal+nerve+fiber+data.">Hierarchical models for epidermal nerve fiber data.</a> Statistics in Medicine. 37 (3), 357-374 (2018).</li><li>Ramshanker, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Prednisolone+on+Serum+and+Tissue+Fluid+IGF-I+Receptor+Activation+and+Post-Receptor+Signaling+in+Humans.">Effects of Prednisolone on Serum and Tissue Fluid IGF-I Receptor Activation and Post-Receptor Signaling in Humans.</a> The Journal of Clinical Endocrinology &amp; Metabolism. 102 (11), 4031-4040 (2017).</li><li>Bouma, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCL20+neutralization+by+a+monoclonal+antibody+in+healthy+subjects+selectively+inhibits+recruitment+of+CCR6++cells+in+an+experimental+suction+blister.">CCL20 neutralization by a monoclonal antibody in healthy subjects selectively inhibits recruitment of CCR6+ cells in an experimental suction blister.</a> British Journal of Clinical Pharmacology. 83 (9), 1976-1990 (2017).</li></ol>

The authors have nothing to disclose.

This manuscript describes a practical procedure for the study of human immune memory responses in vivo, using cutaneous antigen recall and cell harvest by suction blister induction. TST was used as an example of intradermal antigen deposition and BCG-vaccine recall. Finally, an example of SB specimen characterization by flow cytometric and multiplex cytokine analysis was provided, demonstrating that roughly a third of the SB cells were antigen-specific polyfunctional T-cells of the effector memory phenotype.
The critical steps of this protocol include the intradermal injection technique, the suction blister induction, and the blister puncture. Firstly, a correct intradermal deposition requires trained personnel. An incorrect deposition can lead to suboptimal results. PPD is generally a well-known and safe cutaneous antigen, but its composition may vary between manufacturers, limiting comparability13,20. This report provides instructions for a single intradermal deposition of 2 TU, and optionally two parallel depositions (2 x 2 TU), which may require additional ethical approval. However, other studies have used 10 TU, which increase the likelihood of a strong skin reaction10,21. During the SB induction step, small stepwise increases in the negative pressure will reduce the risk of hemorrhage and blister rupture. The chances of contamination with red blood cells or leucocytes from the bloodstream are generally low2. The aseptic puncture technique prevents microbial contamination and avoiding contact between the puncture needle and the dermal blister floor reduces impurities of debris or resident skin cells. However, some researchers prefer to harvest SB fluid by applying a rolling pressure to the punctured blister10. It may be necessary to puncture the septa within the blister. The SB technique itself is minimally invasive; collapsed SBs heal without scarring and infections are very rare.2 However, some degree of hypo-pigmentation may occur and SB induction should probably be avoided in people with a history of colloid scarring.
Technical limitations include low total cell yields and consequently limited options for long-term storage. Relationships between the leucocyte yield, clinical TST responses, the blister size, and erythrocyte contamination has been thoroughly described elsewhere2,10. In the BCG-recall experiments presented here (Figure 1), the median total yield from each blister was 50,000, implying a small-scale experimental set-up using these cells, especially if SBCs are studied alone15. However, the specific T-cell population in an SB sample mostly exceeds what is found in PBMC samples in both relative and absolute cell counts. In the example presented in Figure 2, the number of triple-positive PPD-specific T-cells in a sample of 100,000 SB cells were more than 2x greater than the number found in 1 x 106 PBMCs from the same donor (data not shown). A visual scoring of the TST reaction is the most common clinical method for the evaluation of M. tuberculosis memory. Of note, the underlying adaptive immunological reaction does not peak at the same time as the clinical skin reaction2,21. Not all BCG-vaccinated or Mtb-exposed individuals will develop strong TST reactions and strategies for classification, and a handling of samples from TST non-responders, as well as a preexisting mycobacterial sensitization in the study population, need to be considered before initiating a recall trial using this method. Also, in both SB sampling and visual scoring, there is a potential for a theoretical bias in individuals with reduced skin responses due to global or skin-related impaired immunity as seen, for instance, in HIV-infection and certain age groups2,13,18,20. In addition, an immunological boosting of the TST reaction with repeated testing is well known20,22. However, the SB cell phenotypes remain rather constant when TSTs are repeated10. This observation supports the role of SB recall in longitudinal studies of cellular immune responses.
For T-cell immunologists, SB recall allows for the harvest of high fractions of antigen-specific cells. However, the timing of SB for a sampling from a PPD-deposition is critical as both the cellular composition and the cytokine microenvironment change over time. In this protocol, blisters were induced 7-day post-challenge and the isolated cells were primarily CD4+ effector memory T-cells including high fractions of cells with a co-expression of IFN-&#947;, TNF-&#945;, and IL-2. These observations are in line with previous studies describing how T-cell activation, proliferation, and differentiation occurs in PPD-primed skin2,15,21. Kinetic SB studies in PPD-sensitized individuals suggest that the very early skin response is unspecific; however, already within the first days following the PPD-challenge, the response becomes dominated by CD4+ T-cells (both central memory and effector memory phenotypes have been described) and, after 3 days, the response is dominated by high fractions of PPD-specific cytokine producing CD4+ T-cells2,8,10,15,21. This adaptive cytokine response remains detectable for more than 2 weeks2,8,10,23. Day 7 post-TST appears to be the most optimal time point for the collection of cytokine producing memory CD4+ T-cells2. However, other time points may, of course, be relevant depending on the antigen and response of interest. Of note, in one study, the adaptive PPD response has been reported to peak a little earlier with a decrease in the pro-inflammatory cytokine secretion already at 5 days post-TST10.
SB fluid contains high levels of both pro-inflammatory cytokines and other proteins shown to be representative of the skin microenvironment15,24. Kinetics studies have shown that TNF-&#945; and IFN-&#947; levels in SB fluid peak after 3 days while IL-2 levels peak after 7 days2,10, probably reflecting the dominance of adaptive responses at this later stage. Because SB cells have been activated in vivo, they exhibit a high spontaneous cytokine release as well as a potential for a specific release upon restimulation (Figure 3 and 4).
This report focuses on CD4+ T-cell immunity. As demonstrated in Figure 2, the majority of the isolated T-cells were indeed CD4+, while there were almost no CD8+ T-cells in the suction blister fluid. This is in line with other SB PPD-recall studies reporting low proportions and an inferior migratory capacity of CD8+ T-cells compared to CD4+ T cells2,8,10,23. A further characterization of the CD8+ contribution would be preferable; however, this is beyond the scope of this report.
T-cells are considered essential for the immune control of Mtb; however, it has been difficult to identify a reliable correlate of the protection reflected in the adaptive immune response from the blood25,26. This roadblock severely hinders the development of new TB vaccines, as there currently is no alternative to large and very costly efficacy trials27. TB vaccine developers determine vaccine immunogenicity by assessing small and transient changes in the vaccine-specific T-cell populations in the blood28,29. However, it is questionable whether the small fraction of circulating antigen-specific T-cells found in the blood is relevant (i.e., capable of migrating to the site of an infection and representative of the T-cell-rich microenvironment controlling Mtb)27,30,31.
Based on previous studies and the data presented herein, the SB cutaneous recall model represents an untapped potential for the study of vaccine-specific T-cells. Not only does the model enable a recall of a vaccine response generated decades ago, it also allows the evaluation of the true memory potential in a tissue-specific context15,18,19,21. Novel, specific skin tests, which include antigens also comprised of candidate subunit TB vaccines, suggest new opportunities for vaccine evaluation using this model28,32. Furthermore, transcriptomic analyses suggest that a cell-mediated immune response generated in PPD-challenged skin is similar to the response found in the Mtb-infected lung18.
While skin punch biopsies also allow for a cell harvest from the skin and provide spatial information, compared with SB sampling, the method is more invasive and requires enzymatic or mechanic processing to prepare single-cell suspensions33. Measurements of cytokines and cell markers are comparable between the two methods2,10.
The suction blister method has already been applied in many areas of medical research besides dermatology, either alone or in combination with a systemic or local skin challenge. Examples include studies of sepsis, Epstein-Barr virus-associated lymphoproliferative disease, diabetic neuropathy, glucocorticoid intake effects, and human trials testing therapeutic antibodies or models for T-cell-targeted therapies10,34,35,36,37,38.
From a therapeutic point of view, the SB cutaneous recall method offers unique advantages to study the central T-cell memory potential and-from both a biological and technical point of view-the skin seems to provide a relevant sampling compartment2,15,18,19,21. In particular, compared to traditional, passive sampling of circulating PBMCs, the SB cutaneous recall method allows for the study of T-cells that have proven the ability to migrate from the lymph node in response to their relevant antigen, and complete the local expansion and differentiation in a tissue-specific in vivo context15,18,19,21.
In conclusion, the model demonstrated here could be relevant for researchers within the field of human adaptive immunity and T-cell targeting agents (i.e., in infectious disease vaccinology or cancer research). The TST model applied in this protocol is, of course, of special relevance in the field of TB vaccinology. However, the basic concept of this model is highly applicable in other fields of research.

A skin SB is an artificially induced blister, which allows for the harvest of cells and fluid directly from the skin. The technique of raising SBs by vacuum is a well-known tool within the field of dermatology used for the study of skin immunology in health and disease1,2,3,4,5,6,7,8. This report demonstrates how the SB technique combined with cutaneous antigenic recall (SB cutaneous recall method) can provide direct insights into adaptive immune responses in vivo.
The principle behind the SB induction is simple: a light vacuum is applied to a small area of the skin. The negative pressure will eventually force the epidermis to separate from the dermis, creating a local blister filled with fluid and cells1,2,9. The blister fluid can be harvested by a fine needle aspiration and the content can be used for further study in vitro.
In recent years, there has been an increasing interest in the SB method for the study of in vivo immune responses other than diseases restricted to the skin10,11. The study of the adaptive immunity in humans is limited by the fact that the cells and cytokines of interest are sampled from peripheral blood because an invasive sampling of the lymph node or gastrointestinal mucosal tissue may be unacceptable and unethical. An example is the study of long-lived human memory T-cell responses after vaccination12. In such trials, the sampling of relevant T-cells can be a great challenge, because the relevant population of cells that mediate immunity resides in the lymphoid tissue, and only a very limited number of specific T-cells circulate in the peripheral blood.
The SB technique offers a unique opportunity to the study of memory T-cells and other specific cell populations. Following the cutaneous inoculation of antigen, T-cells specific for the antigen are recruited from their lymphoid hideaways to the skin and can easily be sampled from the SB. The methodology and research applications of this cutaneous recall model were described by Akbar et al. in 20132. A commonly used antigen for skin recall is the commercially available purified protein derivative (PPD) of mycobacteria used to perform a tuberculin skin test (TST)13, where PPD is injected into the dermal layer of the skin. In individuals with an existing immunological memory towards PPD [e.g., individuals with an Mycobacterium tuberculosis (Mtb) infection or a prior Bacillus Calmette-Gu&#233;rin (BCG) vaccination], the antigen deposition results in a recall response with migration to the skin and an in vivo clonal expansion of PPD-specific CD4+ T-cells2,11,14,15.&#160;As a result, high fractions of PPD-specific polyfunctional CD4+ T-cells accumulate in the skin ready for SB sampling. T-cells collected by this method have proven to be robust and are sufficiently abundant to be characterized by a range of immunoassays and by a long-term in vitro culture15. Thus, the cutaneous recall model and the SB induction may prove a valuable method to study in vivo T-cell responses by ex vivo analyses, and increased knowledge of this approach may benefit researchers with interests in cellular immunology and vaccinology.
This report provides the first stepwise guide on how to induce human skin suction blisters in PPD-injected skin. The cutaneous antigen recall model is demonstrated using the TST in BCG-vaccinated volunteers. Finally, the relevant ex vivo analysis of the cells and cytokines isolated by SB is exemplified. Phenotypical and functional characteristics of PPD-specific T-cells obtained by the SB method are thoroughly described elsewhere2,10,11,16,17,18,19. This report aims to discuss the practical and immunological aspects of the methodology to ease the application of this technique by other research groups.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>Gloves for laboratory use</td>
			<td>Imtex, Denmark</td>
			<td>1013</td>
			<td></td>
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			<td>Desinfection spray&#160;</td>
			<td>Apotek, Denmark</td>
			<td></td>
			<td>82% alcohol, with glycerin, for human skin&#160;</td>
		</tr>
		<tr>
			<td>Alcohol swaps</td>
			<td>Mediq, Denmark</td>
			<td>1131892</td>
			<td></td>
		</tr>
		<tr>
			<td>PPD RT 23 &#34;SSI&#34; (2 T.U./0.1ml)</td>
			<td>AJ Vaccines</td>
			<td></td>
			<td></td>
		</tr>
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			<td>&#160;Myjector syringe with fixed needle&#160;</td>
			<td>Terumo</td>
			<td>A236</td>
			<td>1 ml/29G/12mm</td>
		</tr>
		<tr>
			<td>Ruler&#160;</td>
			<td>not relevant</td>
			<td></td>
			<td>&#160;for skin reaction evaluation</td>
		</tr>
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			<td>Ballpoint pen</td>
			<td>not relevant</td>
			<td></td>
			<td>&#160;for skin reaction evaluation</td>
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			<td>Portable Lung Suction Unit</td>
			<td>Laerdal Medical, Denmark</td>
			<td>78000011</td>
			<td>NOTE.&#160; Modified for ranges below -40kPa and supplied with pressure gauge&#160; (ref. 1031107) by the Technical Dept. , Hvidovre Hospital</td>
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			<td>Negative Pressure Chamber Assembly, unheated with connecting tubing</td>
			<td>Electronic Diversities, Finksburg, MD, USA</td>
			<td></td>
			<td></td>
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		<tr>
			<td>Orifice Plate, 47mm, 10mm Hole</td>
			<td>Electronic Diversities, Finksburg, MD, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Negative Pressure Instrument Seal Kit</td>
			<td>Electronic Diversities, Finksburg, MD, USA</td>
			<td></td>
			<td></td>
		</tr>
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			<td>Negative pressure Chamber Strap&#160;</td>
			<td>Electronic Diversities, Finksburg, MD, USA</td>
			<td></td>
			<td>12 inches</td>
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		<tr>
			<td>Micropore Surgical tape</td>
			<td>&#160;3M</td>
			<td>1533-1</td>
			<td>2.5cm x 9.1m</td>
		</tr>
		<tr>
			<td>Cap from 15ml plastic tube</td>
			<td>Sarstedt, Germany</td>
			<td>62,547,254</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubigrip bandage</td>
			<td>M&#246;lnlycke Health Care, Sweden</td>
			<td>1443</td>
			<td></td>
		</tr>
		<tr>
			<td>Cosmopor steril adhesive dressing</td>
			<td>Hartmann</td>
			<td>9008337</td>
			<td>7.2 x 5 cm</td>
		</tr>
		<tr>
			<td>2ml Syringe Concentric Luer Slip&#160;</td>
			<td>Becton Dickinson&#160;</td>
			<td>300185</td>
			<td></td>
		</tr>
		<tr>
			<td>Microlance 23G/30mm needle</td>
			<td>Becton Dickinson</td>
			<td>300700</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-Lock microcentrifuge tubes (autoclaved)</td>
			<td>Eppendorf</td>
			<td>&#160;0030120086</td>
			<td>Autoclaved</td>
		</tr>
		<tr>
			<td>Minispin plus centrifuge</td>
			<td>Eppendorf</td>
			<td>5453000011</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco AIM V Medium</td>
			<td>Life Technologies</td>
			<td>12055-091</td>
			<td></td>
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</table>

a suction blister protocol to study human t-cell recall responses in vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

Eight healthy adult volunteers (median age: 30 years, range: 26 - 43 years) with a documented previous BCG vaccination (median time from BCG-vaccination: 5.5 years, range: 1 - 30 years) were included. The participants were challenged intradermally with 2 TU of PPD followed by a TST evaluation on day 3. SBs were induced on day 7 and harvested on day 8, and all blisters were raised using suction blister chambers with 10 mm orifice diameters. Seven individuals were given 2 separate PPD inoculations simultaneously in the same arm followed by 2 parallel SB inductions (please refer to the note regarding multiple PPD depositions below step 1.4 in the Protocol). Peripheral blood was drawn on day 7 for plasma and a PBMCs isolation by density gradient centrifugation. Plasma and fluid supernatants from SBs were stored at -20 &#176;C. Fresh SB cells (SBCs) were counted using nigrosine stain and microscopy.
The clinical TST responses and SBC yield are presented in Figure 1. The median size of the TST indurations was 10.25 mm (range: 0 - 20 mm) and the median cell number per blister was 50,000 (range: 15,000 - 210,000 cells, number of blisters: 15). Two of the volunteers had no clinical response in either of the 2 TSTs and a corresponding low total cell yield of 15,000 cells/blister. The cell yield was associated with the mean clinical response of TST (Spearman&#39;s r = 0.643, p = 0.094).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57554/57554fig1.jpg" /> 
Figure 1: Suction blister cell yield. (a) This panel shows a representative microscopy of nigrosine-stained cells isolated from SBs raised 7 days post-TST. (b) This panel shows relationships between the mean TST induration (mm) and the mean cell yield (n/blister) in 8 BCG-vaccinated volunteers (number of blisters = 15). The dots represent individual mean measurements. Please note that 2 dots are overlapping as 2 volunteers both had a TST induration of 0 mm and a cell yield of 15,000. <a href="https://www.jove.com/files/ftp_upload/57554/57554fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To demonstrate the flow cytometric SB characterization, SBCs were obtained from a 43-year-old volunteer who had been BCG-vaccinated 30 years earlier and had no known exposure to Mtb. The SBCs were isolated following the induction of a single blister (induration: 1.4 mm/100,000 SBCs). Figure 2 shows representative plots of the intracellular cytokine staining of the SBCs versus PBMCs.
In this volunteer, the fraction of CD3+CD4+CD8- SBCs increased from 67% in unstimulated cells to &#62; 90% upon a PPD in vitro restimulation, whereas the fraction of CD3+CD4+CD8- PBMCs remained constant (~ 51%, Figure 2). Over 92% of the in vitro PPD-, as well as the unstimulated CD3+CD4+CD8- SBCs, were of the effector memory type (CCR7-CD45RA-, data not shown). The overall fractions of specific CD3+CD4+CD8- cells induced by PPD-stimulation were higher in the SBCs compared to the PBMCs (33.1 vs. 0.2%, unstimulated samples subtracted). In the PBMCs, the fractions of polyfunctional PPD-specific CD3+CD4+CD8- cells were all &#60; 0.05%.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57554/57554fig2.jpg" /> 
Figure 2: Representative flow cytometry plots of SBCs versus PBMCs. Panels a and b show representative density plots of CD8+ and CD4+ populations in (a) unstimulated vs. PPD-stimulated PBMCs and (c) SBCs. Panels b and d slow plots of intracellular cytokine staining in PPD-stimulated CD3+CD4+CD8- cells for (b) PBMCs and (d) SBCs. <a href="https://www.jove.com/files/ftp_upload/57554/57554fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
As expected, CD3+CD4+CD8- SBCs were activated in vivo, illustrated by a high proportion of the cells staining for upregulated cytokines (12%), with the predominant cytokines being TNF-&#945; and IFN-&#947;. However, upon PPD-stimulation, the cytokine secreting cells shifted towards a triple- or double-positive IFN-&#947;+TNF-&#945;+IL-2+ (17%) and IFN-&#947;+TNF-&#945;+ (15%) profile (Figure 3b, PBMC profiles from the same donor are included for comparison in Figure 3a). The cytokine expression profiles for PPD-stimulated effector memory CD4+ T-cell subsets (CD3+CD4+CD8-CCR7-CD45RA-) were comparable to the CD3+CD4+CD8- population presented in Figures 2 and 3 (data not shown).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57554/57554fig3.jpg" /> 
Figure 3: Cytokine profiles for CD4+ PPD-stimulated and unstimulated SBCs versus PBMCs.&#160;These panels show individual profiles for cytokine producing (a) CD3+CD4+CD8- PBMCs cells and (b)&#160;CD3+CD4+CD8- SBCs. The bars represent fractions of antigen-specific cytokine profiles in unstimulated cells (white bars) and upon an in vitro stimulation with PPD (colored bars).&#160;
To explore the SB fluid as a source of information on the cytokine microenvironment at the site of the skin testing, the cytokine levels were measured in fluid harvested from SBs induced 7 days post-TST (Figure 4a). The median levels of IFN-&#947;, TNF-&#945;, and IL-2 were 339 pg/mL, 19 pg/mL, and 1 pg/mL, respectively (n = 6). The plasma levels were generally very low. Cells isolated from SBs produced high levels of pro-inflammatory cytokines ex vivo (Figure 4b). Titrations of the SBCs from 1 volunteer were cultured for 4 days in the presence of virulent M. tuberculosis &#177; autologous PBMCs. The IFN-&#947; levels were &#62; 30-fold higher in cultures containing SBCs, irrespective of the presence of PBMCs.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57554/57554fig4.jpg" /> 
Figure 4: Cytokine levels in SB fluid, plasma, and Mtb-infected culture supernatants. (a) This panel shows cytokine levels in SB fluid supernatants and plasma from 6 BCG-vaccinated volunteers. The bars represent the median cytokine levels; the error bars represent the range. (b) This panel shows the cytokine levels in supernatants from 4 parallel 600 &#181;l ex vivo 4-day cultures of 1 x 106 PBMCs (black bars), 1.75 x 105 SBCs (white bars), and 1 x 106 PBMCs spiked with either 0.5 or 1.75 x 105 SBCs (grey bars) infected with Mtb.&#160;<a href="https://www.jove.com/files/ftp_upload/57554/57554fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo at JoVE.com

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

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11.9K Views.  Statens Serum Institut. Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

Video: A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...