The authors are funded by the Wellcome Trust through the Edinburgh Clinical Academic Track (C.J.C.J.) and Programme Grant (Y.H., and R.M.M.) and Fellowship (J.R.G) funding, the BBSRC (G.C.), the American Asthma Foundation (E.R., H.J.McS. and R.M.M.), Asthma UK (H.J.McS.) and the Rainin Foundation (D.J.S. and R.M.M.).

NOTE: All procedures in this protocol are performed in accordance with guidelines set out by the United Kingdom Home Office and the University of Edinburgh Veterinary Services.
1. Infection of Mice by Gavage
<ol>
	<li>Store Heligmosomoides polygyrus L3 larvae in distilled water for up to six months at 4 &#176;C.</li>
	<li>Before use, wash L3 larvae three times in distilled water: centrifuge at 300 x g for 10 min (with brake), remove all but 500 &#956;l of water (to avoid disturbing pelleted L3 larvae) and resuspend the pellet each time.</li>
	<li>For the third wash, add water to an exact volume (typically 40 ml) and aspirate 20 &#181;l with a 200 &#181;l tip cut to widen its aperture. Place two 20 &#181;l samples on the surface of a 60 mm culture dish and count the L3 larvae (usually mobile, and best viewed under 50X magnification with a dissecting microscope). Resuspend the final pellet in distilled water to a concentration of 2,000 L3 larvae per ml.</li>
	<li>For life cycle production, infect 8 week-old F1 (C57BL/6xCBA) mice with 400 H. polygyrus L3 larvae in 200 &#956;l of distilled water by oral gavage (restrain mice in the upright position by the scruff of the neck and gently pass the blunt gavage needle through the mouth and esophagus into the stomach). Agitate thoroughly prior to each infection (larvae settle quickly in water) and aspirate 200 &#181;l in a 1 ml syringe; use a dedicated gavage needle with a rounded end. For experimental infection of younger mice (6-8 weeks old), or other inbred strains (e.g. C57BL/6 or BALBc), infect mice with 200 L3 larvae.</li>
</ol>
2. Propagation and Maintenance of H. polygyrus
<ol>
	<li>Place charcoal in the center of a large plastic tub and allow cold tap water to run over it for a minimum of 30 min (unwashed activated charcoal is toxic to L3 larvae). Drain the water from the tub and place the charcoal on two layers of absorbent paper, leaving it exposed to room air until completely dry.</li>
	<li>If eggs are required, scrape feces first out of the colon with forceps (and scissors if necessary). If a large number of L3 larvae is needed, place mice on a wire grid and collect fecal pellets over several days.</li>
	<li>Mix the feces with granulated charcoal at a ratio of at least a 1:1, to achieve a consistency just damp enough to adhere to filter paper. Smear a thin layer on the center of some dampened filter paper in a petri dish and place this in a humid box (add some damp paper towel and a dish of water) in the dark for 12-14 days.</li>
	<li>Remove L3 larvae from day 7 onwards, and collect them on at least two occasions before the paper is discarded. The larvae form a ring around the edge of the filter paper; lift the filter paper out of the petri dish and rinse the larvae that are left of the plate (using a pipette and 5 ml of sterile water per plate) into a 50 ml tube.</li>
	<li>Lift off the filter paper and harvest the remaining larvae left on the plate with distilled water into a 50 ml tube. Centrifuge the effluent solution at 300 x g for 10 min. Wash the larvae three times with distilled water and then store at 4 &#176;C in up to 50 ml of distilled water until required. 
	NOTE: Larvae remain viable and infective for at least 6 months.</li>
</ol>
3. Collection of Adult H. polygyrus Worms
<ol>
	<li>Prepare the modified Baermann Apparatus in advance as shown in Figure 1.</li>
	<li>Cull mice fourteen days after infection.</li>
	<li>Wash the abdomen with 70% ethanol. Cut the skin over the abdomen and pull back to reveal the anterior abdominal wall. Make midline incision to enter the peritoneal cavity.</li>
	<li>Remove the entire small and large intestine (from proximal duodenum to distal rectum). Place into a dry Petri dish.</li>
	<li>Straighten the gut along its entire length; excise the feces-containing colon; place this into a separate dish for egg preparations later.</li>
	<li>Excise the proximal 20 cm of small intestine that contains the adult worms &#8211;identified by the relatively thick wall of the duodenum and often a red appearance due to the intra-luminal worms. Place into a (100 mm diameter) Petri dish with 5 ml of Hanks&#8217; Solution, warmed to 37 &#176;C (two specimens per dish).</li>
	<li>Open the worm-filled proximal gut portion longitudinally with scissors (round-ended scissors are best for this), and scrape down inside of gut lining with two glass slides to remove the worms. Then discard the clean gut wall.</li>
	<li>Tip worms into small muslin bags, staple closed and secure with paperclips around the edge of the glass funnel (Figure 1).</li>
	<li>Fill funnel with Hanks&#8217; Solution and add approximately 4 Petri dishes of worms into each funnel.</li>
	<li>Place apparatus in 37 &#176;C incubator for 1-2 hr, gently agitating half way through to dislodge debris from the gut preparation that may occlude the muslin filter. Take care to avoid spillage of debris outside the muslin bag &#8211; this will cause contamination of the final HES preparation. Adult worms should have slowly migrated through the muslin cloth and settled at the bottom of the glass test tube. Carefully detach the test tube from the connecting rubber hose over the sink (taking care to avoid losing worms at this point).</li>
	<li>Using a plastic pipette, transfer the worms into a 50 ml tube and wash six times with Hanks&#8217; Solution (allow worms to settle with gravity, remove media with a stripette, add 40 ml of Hanks&#8217; Solution and repeat five times). 
	NOTE: worm culture must be kept sterile from this point onwards.
	<ol>
		<li>Move to a laminar flow hood and wash another six times in sterile Hanks&#8217; Solution supplemented with 100 U/ml penicillin and 100 &#181;g/ml streptomycin, ready for in vitro culture.</li>
	</ol>
	</li>
	<li>Count the adult worms recovered by taking two samples of 20 &#181;l taken up with a yellow tip cut to widen its aperture; expect approximately 50% of the quantity of inoculated larvae.</li>
</ol>
4. Setting Up Cultures for HES: Medium Preparation, Washing Adult Worms
<ol>
	<li>Soak the worms from 3.11 in approximately 10 ml of RPMI supplemented with 10% Gentamycin for 20 min, leaving the tube resting at an angle to ensure worms are fully covered.</li>
	<li>Perform this in a laminar flow hood: wash again six times with Hanks&#39; Solution (supplemented with 5 U/ml penicillin and 5 &#956;g/ml streptomycin).</li>
	<li>Prepare H. polygrus media.</li>
	<li>Maintaining sterility in a laminar flow hood, to 500 ml of RPMI1640, add 11.1 ml of 45% glucose (final concentration 1.2% as RPMI1640 contains 0.2% glucose), 5 ml of 100x Peniclllin-Streptomycin (final concentration 5 U/ml penicillin, 5 &#956;g/ml streptomycin), 5 ml of L-glutamine (final concentration 2 mM), and 5 ml of Gentamycin (final concentration 1%). Do not add FCS.</li>
	<li>Aliquot worms into vented T25 flasks, approx. 1,000 worms in 15 ml H. polygyrus media per flask, and place upright in 37 &#176;C incubator (5% CO2) for 3 weeks.</li>
</ol>
5. Preparation of HES
<ol>
	<li>Collect HES-containing culture media from cultures at intervals of no longer than twice per week, setting aside the first collection after 24 hr of culture (due to high levels of LPS contamination and host proteins &#8211; can be processed separately or discarded). Keep each collection separate and clearly labeled with the date and batch number. Replace with an equal volume of H. polygyrus media on each occasion.</li>
	<li>Centrifuge HES-containing media at 400 x g for 5 min. Then filter sterilize through 0.2 &#181;m low-protein-binding filters into 50 ml tubes. Store in the -20 &#176;C freezer clearly labeled with date of worm harvest and date of HES collection.</li>
	<li>After 21 days of HES collection from culture, discard worms.</li>
	<li>Pool 500-1,000 ml of HES supernatant (usually from frozen stock, and not including the first 24 hr collection) and concentrate over a 3,000 MWCO filter in the ultrafiltration device under nitrogen pressure. 
	NOTE: Be very careful not to let the filter run dry.
	<ol>
		<li>To set up the filter device, first wash the 3 kDa membrane shiny side down in a 1 liter beaker with distilled water for 3 x 20 min whilst stirring. Assemble the ultrafiltration device as per manufacturer&#8217;s instructions with filter membrane shiny side up. Place in cabinet at 4 &#176;C and pass 50 ml of distilled water through before starting to concentrate pooled HES.</li>
		<li>Add each tube of HES into the filtration device as required (100-140 ml per day), until the volume is concentrated down to 2-5 ml.</li>
	</ol>
	</li>
	<li>In order to remove contaminants from the HES-containing culture media, add 50 ml of pyrogen-free PBS to the filtration device and and then concentrate down to approximately 2 ml. Repeat this step twice (150 ml of PBS in total). Transfer HES into a 15 ml tube, filter sterilize (with a 0.2 &#956;m filter) in a laminar flow hood and measure protein concentration using a spectrophotometer (E280 = 10) or by Bradford assay.</li>
	<li>Aliquot, label with batch number and date, and freeze at -80 &#176;C.</li>
	<li>Perform a chromogenic LAL assay according to the manufacturer&#8217;s protocols on each batch of HES prior to use. If LPS levels are greater than 1 U LPS per 1 &#181;g protein, consider not using this batch for in vivo experiments or in vitro cultures.</li>
	<li>Process the HES collected at 24 hr separately in the same manner; it will contain LPS and some host proteins and, while not suitable for functional experiments, it is a useful source of individual molecules that may be isolated by monoclonal antibody affinity purification.</li>
</ol>

The authors have no conflicts of interest.

The life cycle of H. polygyrus proceeds in a reliably consistent fashion. Following natural ingestion or oral gavage of L3 larvae on day 0, cysts begin to form under the duodenal serosa by day 5, progressing to larval moults and then emerging as adult worms into the gut lumen from day 10, eggs can be seen in feces from day 14 and granulomas are visible on the duodenal serosal surface from day 21. The protocol described above (and summarized in Figure 6) allows for high-throughput production of H. polygyrus excretory-secretory products (HES), in addition to reliable recovery of viable L3 larvae for future experimental and life cycle infections.
H. polygyrus&#160;infection has been shown to be protective in models of asthma dependent on OVA or Der p 1 (House dust mite allergen)14. Furthermore, suppression of airway inflammation could be transferred with CD4+CD25+ regulatory T cells 14&#160;or CD19+CD23hi regulatory B cells 24 from non-sensitised,&#160;H. polygyrus-infected mice. In models of autoimmunity, H. polygyrus&#160;infection has been shown to be suppressive in the&#160;experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis 9, and suppression can be transferred with either CD4+ T cells or CD19+ B cells from infected mice 24.
Heligmosomoides polygyrus excretory-secretory products (HES) modulate the host immune response and suppress Th2-mediated inflammation by a number of mechanisms (outlined in Figure 7), including: a) Inhibition of dendritic cell responsiveness to stimulation 25, b) induction of CD4+Foxp3+ regulatory T cells 18 . and c) blockade of IL-33 production 20. HES has been shown to be protective when administered at sensitization or challenge in the OVA-alum model of asthma 19 and also when administered intranasally with Alternaria extract allergen 20, through suppression of early IL-33 release. Avoidance of LPS contamination of HES is often crucial for the success of future immunological experiments. In the protocol outlined here, the critical steps in achieving this are to ensure that debris contained within the muslin bags of the Baermann Apparatus does not enter the final HES preparation (see 2.10) and to set aside ES solution from the first 24 hr of culture (see 5.1).
Over recent years, HES has been thoroughly characterized at the proteomic level with over 370 distinct proteins identified 26,27. In addition, ES from the 4th stage larvae has been subjected to proteomic analysis 28. Ongoing work includes characterizing the major glycan components of HES, known to be strongly immunogenic 3, and secreted micro-RNAs that are encapsulated in 50-100 nm vesicles or exosomes (Buck et al., submitted for publication). With the establishment of reproducible protocols for the collection of ES from this highly immunomodulatory parasite, and with extensive proteomic data identifying the molecular components of HES, the stage is now set for the identification and therapeutic testing of individual molecules from H. polygyrus that can mediate key effects on the host immune system.

Heligmosomoides polygyrus is a natural murine gastrointestinal helminth that is closely related to highly-prevalent human nematode parasites 1. In contrast to other nematode models such as Nippostrongylus brasiliensis, H. polygyrus consistently establishes chronic infection in mice as a direct result of multiple powerful immunomodulatory mechanisms it employs to suppress the host immune response 2.
H. polygyrus has a direct life cycle: infective L3 larvae are ingested by feco-oral transmission (or administered by oral gavage in the laboratory setting), whereupon they migrate to the subserosal layer of the duodenum and encyst before returning to the intestinal lumen as adult worms approximately eight days after initial infection. Mating and egg production occurs by day 10 and it is possible to harvest adult worms for culture and collection of excretory-secretory products from day 14 onwards 3. H. polygyrus also interacts with the commensal microflora, with increased Lactobacilli present in infected susceptible mice 4-6, and increased levels of H. polygyrus infection following exposure of mice to Lactobacilli 6.
Active infection with H. polygyrus has been shown to protect against immunopathology in many animal models of autoimmunity 7-10, colitis 11,12 and allergy 13-16. There has consequently been great interest in the potential of Excretory-Secretory molecules from this parasite (&#8220;HES&#8221;) to down-modulate pathology in vivo 17,18. Indeed, protective effects are seen following treatment of mice with HES products 19 through pathways which are now being identified 18,20. Here we describe a protocol for the reliable production of Heligmosomoides polygyrus and recovery of its secreted products that can be further utilized for a range of functional biochemical and immunological investigations.

<table><tbody><tr><td>Amicon concentrator</td><td>Millipore</td><td>5112</td><td>Model 8050 50 ml&lt;br/&gt; http://www.emdmillipore.com/INTL/en/product/Stirred-Cell-Model-8050,-50%C2%A0mL,MM&lt;br/&gt; _NF-5122</td></tr><tr><td>Hanks&amp;rsquo; buffered salt solution</td><td>Sigma</td><td>H9394</td><td>500 ml&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/sigma/h9394?lang=en&amp;amp;region=GB</td></tr><tr><td>Penicillin streptomycin</td><td>Invitrogen</td><td>15070-063</td><td>100 ml&lt;br/&gt; http://www.lifetechnologies.com/order/catalog/product/15070063</td></tr><tr><td>L-glutamine</td><td>Invitrogen</td><td>25030-081</td><td>200 MM 100ml&lt;br/&gt; https://www.lifetechnologies.com/order/catalog/product/25030081?ICID=search-25030081</td></tr><tr><td>Activated charcoal</td><td>Merck</td><td>1.09624.0500</td><td>18-35 mesh&lt;br/&gt; http://www.merckmillipore.com/GB/en/product/Charcoal-activated,MDA_CHEM-109624</td></tr><tr><td>Glucose solution</td><td>Sigma</td><td>G8769</td><td>100 ml 45% solution&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/sigma/g8769?lang=en&amp;amp;region=GB</td></tr><tr><td>Falcon tubes</td><td>Sarstedt</td><td>62.547.254</td><td>50 ml&lt;br/&gt; http://www.scribd.com/doc/124320105/Sarstedt-Falcon</td></tr><tr><td>T25 Flasks</td><td>Sigma</td><td>CLS430639&amp;nbsp;</td><td>25cm vented flask&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/sigma/cls430639?lang=en&amp;amp;region=GB</td></tr><tr><td>Chromogenic LAL assay</td><td>Lonza</td><td>50-647U</td><td>QCL-1000 assay&lt;br/&gt; http://www.lonza.com/products-services/pharma-biotech/endotoxin-detection/endotoxin-detection-assays/endpoint-chromogenic-lal-assay.aspx</td></tr><tr><td>Gentamicin</td><td>Gibco</td><td>15710-049</td><td>100 ml&lt;br/&gt; http://www.lifetechnologies.com/order/catalog/product/15710072</td></tr><tr><td>Roswell Park Memorial Institute (RPMI) 1640</td><td>Gibco</td><td>11875093</td><td>500 ml; without L-glutamine&lt;br/&gt; http://www.lifetechnologies.com/order/catalog/product/11875093</td></tr></tbody></table>

cultivation of heligmosomoides polygyrus: an immunomodulatory nematode parasite and its secreted products

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that employs multiple immunomodulatory mechanisms to establish chronic infection in mice and closely resembles prevalent human helminth infections. H. polygyrus has been studied extensively in the field of helminth-derived immune regulation and has been found to potently suppress experimental models of allergy and autoimmunity (both with active infection and isolated secreted products). The protocol described in this paper outlines management of the H. polygyrus life cycle for consistent production of L3 larvae, recovery of adult parasites, and collection of their excretory-secretory products (HES).

Susceptibility to infection with H. polygyrus is controlled to a large degree by the genetic background of the mouse strain (Table 1); C57BL/6 and CBA mice are highly susceptible 21,22. For maintenance of the parasite life cycle, the F1 hybrid between these two strains has been chosen for its ability to withstand much higher worm burdens without morbidity (excessive intestinal epithelial damage) compared to either parental strain. Oral gavage of 400 L3 larvae is used to maintain the life cycle in F1 mice (resulting in adult worm burdens shown in Figure 2), whilst a dose of 200 L3 larvae is generally used for experiments in homozygous inbred strains (e.g., C57BL/6 or BALBc). However, this dosage may need to be reduced depending on environmental co-factors that may differ between animal facilities, such as variations in gut flora.
Batches of HES have proven reproducible efficacy in functional assays and in protein composition; moreover, when supernatants from each successive week of culture were analysed, up to a total of 4 weeks, the protein profiles were found to be very similar (Figure 3). When HES concentration is measured by Bradford assay (see 5.5), the total protein is usually approximately 1 mg/ml (Figure 4).
An alternative method of concentrating HES is with a centrifugal concentrator (eg Vivaspin 3-kDa cut-off membrane), in which samples are spun at up to 4,000 g in a swing-out rotor centrifuge, removing buffer salts and low molecular weight components. Centrifugal concentration is best suited to small processing volumes (1-10 ml) and are limited to a maximum concentration factor of approximately 30x.
When collecting HES, avoidance of contamination is critical. To avoid contamination with host molecules, we discard HES-containing culture media from the first 24 hr after adult worm harvest from the mouse intestine. We also quantify the level of LPS contamination in each batch of HES with a Chromogenic LAL assay (see 5.7). 1 U of LPS equates to ~100 pg LPS and levels below this are considered negligible 23. In our hands, most batches of HES are significantly below this limit, the mean concentration of LPS in HES being 0.23 U/&#181;g (Figure 5). The effects of LPS in in vivo models of pathology (for instance the suppression of asthmatic responses) requires at least 10 ng of LPS 23. Hence in vivo administration of 5 &#181;g HES from a batch with 100 pg LPS/&#181;g HES will include 500 pg LPS, well below the effective concentration where LPS becomes a problem.
<img alt="Figure 1" src="/files/ftp_upload/52412/52412fig1highres.jpg" /> 
Figure 1: Baermann Apparatus Baermann Apparatus setup for collection of adult H. polygyrus (as described in section 2).
<img alt="Figure 2" src="/files/ftp_upload/52412/52412fig2highres.jpg" /> 
Figure 2: Mean Worm Burden 14 Days After Infection with 400 L3 Larvae Mean worm burdens 14 days after infection with 400 L3 larvae. Data points shown are from 19 separate rounds of infection of C57BL/6xCBA mice. Mean &#177; SEM shown.
<img alt="Figure 3" src="/files/ftp_upload/52412/52412fig3highres.jpg" /> 
Figure 3: Protein Profiles of HES From Successive Weeks in Culture SDS-PAGE profile of HES proteins collected in successive weeks of culture.
<img alt="Figure 4" src="/files/ftp_upload/52412/52412fig4highres.jpg" /> 
Figure 4: Final Quantity of HES from 500 ml ES-Containing Media Yield of HES protein from 11 different batches derived from approximately 500 ml of culture supernatant. Mean &#177; SEM shown.
<img alt="Figure 5" src="/files/ftp_upload/52412/52412fig5highres.jpg" /> 
Figure 5: LPS Contamination of HES Levels of LPS contamination in 41 batches of HES measured by the Limulus ameobocyte assay. Median LPS concentration = 86 U per mg of HES.
<img alt="Figure 6" src="/files/ftp_upload/52412/52412fig6highres.jpg" /> 
Figure 6: Animated Schematic of H. polygyrus Life Cycle Summary of key life cycle stages from oral gavage of L3 larvae, through recovery of larvae and adult worms to isolation of HES.
<img alt="Figure 7" src="/files/ftp_upload/52412/52412fig7highres.jpg" /> 
Figure 7: Animated Schematic of HES and its Functions Key immunomodulatory effects of soluble mediators and exosomes contained within HES.
<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Genotype (and background strain)</td>
			<td>Primary Infection Phenotype</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inbred strains</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A/J, CBA, C3H&#160;</td>
			<td>Highly susceptible&#160;</td>
			<td>22,29</td>
		</tr>
		<tr>
			<td>C57BL/6 and C57BL/10&#160;</td>
			<td>Susceptible&#160;</td>
			<td>22,30</td>
		</tr>
		<tr>
			<td>BALB/c, DBA/2, 129/J&#160;</td>
			<td>Intermediate&#160;</td>
			<td>22,31</td>
		</tr>
		<tr>
			<td>NIH, SJL, SWR</td>
			<td>Low susceptibility&#160;</td>
			<td>22,32</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Transgenic for cytokines or cytokine receptors</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-1&#946;-/-</td>
			<td>More susceptible</td>
			<td>33</td>
		</tr>
		<tr>
			<td rowspan="2">IL-1R-/-</td>
			<td rowspan="2">Less susceptible (a); no change in susceptibility but increased granulomas (b)</td>
			<td>(a) 33</td>
		</tr>
		<tr>
			<td>(b) 34</td>
		</tr>
		<tr>
			<td>IL-2R&#946; Transgenic (C57BL/6)</td>
			<td>Resistant&#160;</td>
			<td>35</td>
		</tr>
		<tr>
			<td>IL-4-/-</td>
			<td>Higher fecundity</td>
			<td>36</td>
		</tr>
		<tr>
			<td>IL-4R-/- (C57BL/6 or BALB/c)</td>
			<td>Highly susceptible</td>
			<td>22</td>
		</tr>
		<tr>
			<td>IL-6&#8211;/&#8211; (BALB/c or C57BL/6)</td>
			<td>Highly Resistant&#160;</td>
			<td>37</td>
		</tr>
		<tr>
			<td>IL-9 Transgenic (FVB)&#160;</td>
			<td>Resistant&#160;</td>
			<td>38</td>
		</tr>
		<tr>
			<td>IL-21R &#8211;/&#8211; (C57BL/6)</td>
			<td>Deficient Th2, decreased granuloma gormation</td>
			<td>39,40</td>
		</tr>
		<tr>
			<td>IL-25-/- (BALB/c)</td>
			<td>More susceptible</td>
			<td>33</td>
		</tr>
		<tr>
			<td>IL-33R (T1/ST2) -/- (BALB/c)</td>
			<td>No change in susceptibility</td>
			<td>33</td>
		</tr>
		<tr>
			<td>TGF&#946;RIIdn (C57BL/6)</td>
			<td>High Th1, Increased susceptibility</td>
			<td>41,42</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Transgenic for T cell markers</td>
			<td></td>
		</tr>
		<tr>
			<td>CD28&#8211;/&#8211; (BALB/c)&#160;</td>
			<td>Marginally higher fecundity</td>
			<td>43</td>
		</tr>
		<tr>
			<td>CD86 (B7-2)&#8211;/&#8211; (BALB/c)&#160;</td>
			<td>Higher fecundity&#160;</td>
			<td>44</td>
		</tr>
		<tr>
			<td>OX40L&#8211;/&#8211; (BALB/c)&#160;</td>
			<td>Higher fecundity&#160;</td>
			<td>45</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Transgenic for innate Immune loci&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Type 1 interferon receptor (IFNAR)-/- (C57BL/6)&#160;</td>
			<td>Higher fecundity, more granulomas</td>
			<td>34</td>
		</tr>
		<tr>
			<td>MyD88&#8211;/&#8211; (C57BL/6)&#160;</td>
			<td>More resistant, more granulomas</td>
			<td>34</td>
		</tr>
		<tr>
			<td>C-KitW/Wv (WBB6)&#160;</td>
			<td>More susceptible</td>
			<td>46,47</td>
		</tr>
	</tbody>
</table>
Table 1: Primary infections with H. polygyrus in genetically different and gene-targetted mouse strains.

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Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus is a murine nematode with powerful immunomodulatory capabilities that closely resemble those of highly-prevalent human helminth infection. Here we describe a protocol for the long-term maintenance of the H. polygyrus lifecycle.

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that ...

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18.3K Views.  University of Edinburgh. Heligmosomoides polygyrus is a murine nematode with powerful immunomodulatory capabilities that closely resemble those of highly-prevalent human helminth infection. Here we describe a protocol for the long-term maintenance of the H. polygyrus lifecycle.

Video: Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense against pathogens. It is well established that Peyer&#39;s patches (PPs) have an essential role in the mucosal immune network by hosting several effector T and B cell subsets. A certain fraction of these effector cells, follicular T helper (TFH) and germinal center (GC) B cells are professionalized in the regulation of humoral immunity. Hence, the characterization of these cell subsets within PPs in terms of their differentiation program and functional properties can provide important information about mucosal immunity. To this end, an easily applicable, efficient and reproducible method of lymphocyte isolation from PPs would be valuable to researchers. In this study, we aimed to generate an effective method to isolate lymphocytes from mouse PPs with high cell yield. Our approach revealed that initial tissue processing such as the use of digestive reagents and tissue agitation, as well as cell staining conditions and selection of antibody panels, have great influence on the quality and identity of the isolated lymphocytes and on experimental outcomes.
Here, we describe a protocol enabling researchers to efficiently isolate lymphocyte populations from PPs allowing reproducible flow cytometry-based assessment of T and B cell subsets primarily focusing on&#160;TFH and GC B cell subsets.

In this study, we present a novel and effective protocol for the isolation of lymphocytes from Peyer&#39;s Patches (PPs), which can be subsequently used for in vivo and in vitro functional assays as well as flow cytometric studies of follicular T helper and germinal center B cells.

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense ...

Cancer Research

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

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 ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Humanized NOD/SCID/IL2r&#947;null (hu-NSG) Mouse Model for HIV Replication and Latency Studies

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in high demand. With a close genetic and behavioral resemblance to humans, small animals such as the mouse are good candidates for human disease models, through which human-like symptoms and responses can be recapitulated. Further, the mouse genetic background can be altered to accommodate diverse demands. The NOD/SCID/IL2r&#947;null (NSG) mouse is one of the most widely used immunocompromised mouse strains; it allows engraftment with human hematopoietic stem cells and/or human tissues and the subsequent development of a functional human immune system. This is a critical milestone in understanding the prognosis and pathophysiology of human-specific diseases such as HIV/AIDS and aiding the search for a cure. Herein, we report a detailed protocol for generating a humanized NSG mouse model (hu-NSG) by hematopoietic stem cell transplantation into a radiation-conditioned neonatal NSG mouse. The hu-NSG mouse model shows multi-lineage development of transplanted human stem cells and susceptibility to HIV-1 viral infection. It also recapitulates key biological characteristics in response to combinatorial antiretroviral therapy (cART).

This protocol provides a method to establish humanized mice (hu-NSG) via intrahepatic injection of human hematopoietic stem cells into radiation-conditioned neonatal NSG mice. The hu-NSG mouse is susceptible to HIV infection and combinatorial antiretroviral therapy (cART) and serves as a suitable pathophysiological model for HIV replication and latency investigations.

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in ...

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, ...

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.

This protocol describes the use of single chain MHC class I complexes to investigate molecular interactions in human CD8+ T cell activation: generation of engineered antigen presenting cells expressing single chain constructs, culture of human CD8+ T cell clone and T cell activation experiments.

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 million IBD patients. It is generally accepted that a dysregulated immune response to gut bacteria initiates the disease and disrupts the mucosal epithelial barrier. We recently show that gut-homing regulatory T (Treg) cells are a promising therapy for IBD. Accordingly, this article presents a protocol for in vivo augmentation of gut-homing Treg cell induction. In this protocol, dendritic cells are engineered to produce locally high concentrations of two molecules de novo, active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and active vitamin A (retinoic acid or RA). We chose 1,25(OH)2D and RA based on previous findings showing that 1,25(OH)2D can induce the expression of regulatory molecules (e.g., forkhead box P3 and interleukin-10) and that RA can stimulate the expression of gut-homing receptors in T cells. To generate such engineered dendritic cells, we use a lentiviral vector to transduce dendritic cells to overexpress two genes. One gene is the cytochrome P450 family 27 subfamily B member 1 that encodes 25-hydroxyvitamin D 1&#945;-hydroxylase, which physiologically catalyzes the synthesis of 1,25(OH)2D. The other gene is the aldehyde dehydrogenase 1 family member A2 that encodes retinaldehyde dehydrogenase 2, which physiologically catalyzes the synthesis of RA. This protocol can be used for future investigation of gut-homing Treg cells in vivo.

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and the active vitamin A (retinoic acid or RA) de novo.

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 ...

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response against helminth infections, where it plays an important role in the expulsion of the parasite. In a murine model of Nippostrongylus brasiliensis infection, IL-9 is produced mainly by CD4+ T lymphocytes and innate lymphoid cells found in the lung, small intestine, and draining lymph nodes. Given the technical difficulties involved in the intracellular staining of IL-9, as well as the complexity of isolating hematopoietic cells from the small intestine upon infection, there is a pressing need for a comprehensive but straightforward protocol to analyze the expression of IL-9 in different lymphoid and non-lymphoid tissues in this model. The protocol described here outlines the kinetics of IL-9 produced by CD4+ T cells and innate lymphoid cells in the lung and small intestine, the main organs targeted by N. brasiliensis, as well as in the mediastinal and mesenteric lymph nodes, throughout the infection. In addition, it details the number of larvae needed for infection, depending on the cell type and organ of interest. This protocol aims to assist in the standardization of assays to save time and resources by offering the opportunity to focus on the specific cells, organs, and disease stages of interest in the N. brasiliensis infection model.

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

IL-9-expressing T and ILC2 cells are induced during N. brasiliensis infection, yet their characterization has been largely overlooked in the infected intestine due to their low frequency and differential kinetics. This protocol describes the isolation of these cells from different target organs and confirmation of their identity via flow cytometry at different infection stages.

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response ...

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

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Chapters in this video

Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

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

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

Humanized NOD/SCID/IL2rγ^null (hu-NSG) Mouse Model for HIV Replication and Latency Studies

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

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model