The expert animal husbandry provided by Tina Martin and David Albright is gratefully appreciated. This research was supported by grants T32-AI-07285 (H. N.), NIH R25 2GM064133 (T.C.L), 1R03-HD061671-01, R24-AI-059830-06 from the NIH.

1. Animals
X. laevis x X. gilli hybrids LG-6 and LG-15 isogenetic clones 1 are from our breeding colony at the University of Rochester (http://www.urmc.rochester.edu/smd/mbi/xenopus/index.htm). LG-6 and LG-15 share the same heterozygous MHC haplotype (a/c) but differ at minor histocompatibility (H) loci. Progeny from these clones are produced by gynogenesis, in which diploid eggs produced by the female are activated by UV-irradiated sperm (no DNA contribution to the progeny).The use of gloves is facultative. Some people prefer to not wear them because it is more difficult to handle the frogs (slippery) and in fact it appears to make the frogs uncomfortable.
2. Purification of gp96 from 15/0 Tumor (Expresses Both Tumor and Minor H-Ags)
Gp96 purification has been previously described 2, 3. Briefly, gp96 is purified by 50-70% ammonium sulfate fractionation, followed by conA-sepharose and DEAE chromatography. About 20-50 &mu;g of protein can be obtained per 1 mL of tumor tissue. Purity of the preparation is determined by SDS-PAGE and sliver staining.
3. Elicitation and Harvest of Peritoneal Leukocytes (PLs) from Minor H-Ag-disparate LG-6 Frogs
<ol>
 <li>Grow a 25 mL overnight E. Coli culture in a 50 mL conical tube at 37&deg;C with shaking.</li>
 <li>The next day heat kill the bacteria by boiling it for 1 hour.</li>
 <li>Centrifuge the heat killed E. Coli for 15 min at 2,000 rpm (1,500 g) at 4&deg;C.</li>
 <li>Remove the supernatant and resuspend the bacterial pellet in 1/10th the original culture volume (2.5 mL) in APBS. At this point the bacterial culture is ready for use and must be used within 24 hrs.</li>
 <li>Inject intraperitoneally (i.p.) 200 (for a 2 inch frog) or 300 &mu;L (for a 3 inch frog) of the heat-killed bacterial preparation per frog using a 25 gauge 5/8 needle.</li>
 <li>Three days after injection PLs are harvested by intraperitoneal lavage.</li>
 <li>Before PL harvesting, adult frogs are anesthetized by immersion in a 0.1% aqueous solution of tricaine methane sulfonate (TMS, MS-222) buffered with sodium bicarbonate for up to 5 min until all movement ceases (duration depends on size and age). Animals wake within 10-20 min after treatment.</li>
 <li>Disinfect the abdomen of the frog with a small amount of 70% ethanol.</li>
 <li>Inject 5 mL (for a 2 inch frog) or 10 mL (for a 3 inch frog) of sterile APBS pre-warmed at room temperature into the peritoneal cavity using a 18 gauge 1 &frac12; needle. Remove the needle and gently massage the frog for a minute to insure that the injected buffer equilibrates with the fluid in the body cavity.</li>
 <li>Use a new 18 gauge 1 &frac12; needle without a syringe to collect the peritoneal fluid that will drip from the back of the needle into a clean 50 mL conical tube. Make sure to retrieve as much of the initial injected volume as possible. Be careful to avoid blood vessels in the central area of the abdominal region.</li>
 <li>Once PLs are harvested put the frog in a container with shallow water until it is awake at which point it can be placed back in its cage.</li>
</ol>
4. Pulsing and Adoptive Transfer of Lg-6 Pls Into Na&iacute;ve Lg-6 Recipients
<ol>
 <li>Wash the PLs once with cold APBS and centrifuge them at 1,000 rpm (750 g) for 10 min at 4&deg;C.</li>
 <li>Remove the supernatant and resuspend the PLs in APBS.</li>
 <li>Pulse the PLs with gp96 at a concentration of 1 &mu;g gp96 per 5 x 105 PLs.</li>
 <li>Once the appropriate amount of gp96 is added to the PLs, mix them by pipeting and incubate on ice for 1 hour.</li>
 <li>Centrifuge the cells either at 1,000 rpm for 10 min at 4&deg;C or at 14,000 rpm for 1 min to remove any unbound gp96.</li>
 <li>Wash the PLs 3X with cold APBS to ensure there is no residual gp96 left.</li>
 <li>Resuspend the pulsed PLs at a concentration of 5 x 105 PLs per 300 &mu;L.</li>
 <li>Adoptively transfer PLs by i.p. injection using 25 gauge 5/8 needle. Inject 300 &mu;L of pulsed PLs (5 x 105 cells) per animal. </li>
 <li>Frogs need to be primed at least 3 days before continuing with skin graft or tumor challenge experiments.</li>
</ol>
5. Skin Graft Assay
Skin graft rejection is a well-established assay in Xenopus 4, 5, 6. In Xenopus, a recipient rejects donor skin displaying 1 or 2 MHC haplotype mismatches within 18-22 days at 21-22&deg;C. In contrast, skin grafts between LG-6 and LG-15 clones that differ only by minor H-Ags are rejected more slowly (more that 30 days). However, this rejection is accelerated in recipients that have been primed against donor minor H-Ags either by a previous skin graft or by immunization with gp96 purified from the donor. No rejection at all occurs when the donor and the recipient are genetically identical (e.g., cloned or fully inbred animals). Therefore, this simple technique is very powerful for characterizing in vivo immune responses elicited by gp96.
<ol>
 <li>Anesthetize donor frog as described in 3.7. 
 Note: Only minimal precautions need to be taken since Xenopus produce potent anti-microbial peptides (e.g., magainin) in the skin that obviates the need for total aseptic procedures. In fact, treatment of the frog with any disinfectant would be detrimental for the skin. In addition, Xenopus do not have any pathogens that can be transferred to humans. Therefore, the use of gloves is optional. However, all the dissection instruments used need to be autoclaved and all solutions need to be sterile.</li>
 <li>Cut and remove a small (20 mm X 5 mm) piece of ventral skin (abdominal skin which appears silvery due to the presence of irridophore pigmented cells) from a LG-15 donor frog using scissors.</li>
 <li>Place the skin in a Petridish containing APBS and keep it on ice. Manipulate the skin tissue very gently; avoid holding it in between forceps.</li>
 <li>Using a razor or scissors cut individual grafts into 5 mm X 5 mm pieces. Keep the fragments in APBS on ice.</li>
 <li>At this point the donor frog is placed in a container with shallow water until it is awake and then it is placed in water containing antibiotics (Penstrep at concentration of 5 mg/L). The frog is kept in water with antibiotics for two days at which point it is returned in normal water. The small wound at the site where the skin was removed does not need to be sewed and heals within a week.</li>
 <li>Anesthetize the recipient LG-6 frog.</li>
 <li>Make a small incision on the dorsal (back) skin of the recipient and insert the 5 mm X 5 mm graft under the skin with the silvery side up. Be careful not to introduce large air bubbles under the skin because that may lead to displacement or even loss of the graft.</li>
 <li>Make sure you note the scissor and forceps markings on the grafts because those are not counted as graft rejection once the scoring starts.</li>
 <li>24 hours later a part of the overlaying host skin needs to be removed from the graft.</li>
 <li>Anesthetize the recipient LG-6 frog. Handle the animal as described in 5.1.</li>
 <li>Using autoclaved scissors cut out a window around the graft so that the graft can be freely visualized. Be careful not to touch the donor skin or to induce bleeding. Let the frog recover as described in 5.5.</li>
 <li>Start scoring the graft right away by taking a sketch. Skin graft rejection is determined by the percent of destruction of the irridophores on the grafted skin.</li>
 <li>The grafts need to be checked every 2-3 days but the animals do not need to be anesthetized for this. To visualize the grafts, the frogs need to be placed in a petridish under a dissecting microscope.</li>
</ol>
6. Whole-mount Immunohistology of Transplanted Skin
Frog whole-mount immunohistology has been previously described 6. Briefly, the frog is anesthetized and placed under the microscope on a sterile paper towel pre-wet with frog water (the working area is also aseptically prepared). The transplanted skin is then harvested together with a small amount of surrounding host skin and it is stained with antibodies similarly to cell staining for flow cytometry analysis 6. Autoclaved instruments and sterile buffers need to be used. After staining the skin is placed on a microscope slide and gently pressed with a cover slip. At this point the skin can be visualized using a fluorescent microscope for different cell populations that have infiltrated the graft. After the procedure the frog is kept in water with antibiotics as described in section 5.5. and returned in normal water. The small wound left where the skin was removed heals within a week.
7. Characterization of Blood Leukocytes
<ol>
 <li>Before removing blood from the frog, one needs to prepare "glass needles" by pulling sterile Pasteur pipettes over flame. The fine extremity of the pipette is sharpened under the stereomicroscope. The pipette is then connected to an aspiration plastic tube. All instruments such as the forceps and scissors need to be autoclaved before use.</li>
 <li>Also prepare ice cold 10 mL of APBS and heparin solution by adding 50 units heparin per 1 mL of APBS. Keep solution on ice. All solutions used need to be sterile.</li>
 <li>Anesthetize frog as described in 3.7, and follow the same animal handling procedures as described in 5.1. .</li>
 <li>Cut the skin above the posterior foot to expose the dorsal tarsus vein.</li>
 <li>Fill the Pasteur pipette with 1-2 mL of APBS+heparin solution.</li>
 <li>Insert the "glass needle" into the vein and start collecting the blood by slowly aspirating with the mouth. It is important to have APBS+heparin solution in the "glass needle" since this will prevent the blood from clotting and clogging the tip of the needle.</li>
 <li>1 to 2 mL of blood can be obtained from one average sized frog.</li>
 <li>The small incision on the frog's leg does not need to be sewed and the wound heals within one week.</li>
 <li>Centrifuge the blood at 1,000 rpm for 10 min at 4&deg;C.</li>
 <li>Remove the supernatant and wash the cells 1X with 10 mL of cold APBS.</li>
 <li>Overlay 2 mL of blood (1-5 x 106 cells) onto 1 mL of ficoll Histopaque 1.077 (Sigma) pre-warmed at room temperature in order to separate the blood leukocytes from the red blood cells.</li>
 <li>Centrifuge 20 min at 1,000 rpm at room temperature with no brake, and then collect the leukocyte band.</li>
 <li>Wash the cells 2X with APBS by centrifugation at 1,400 rpm for 10 min at 4&deg;C to remove the residual ficoll.</li>
 <li>At this point the cells are ready to be stained with antibodies for flow cytometry analysis, or to be use for in vitro culture assays.</li>
</ol>
8. Tumor Transplantation Assay
<ol>
 <li>About a week before the experiment thaw out a fresh batch of 15/0 tumor cells and make sure that the cells start growing well. Culture medium is succinctly described in the next section and in more detail in 3.</li>
 <li>Expand the 15/0 tumor cells.</li>
 <li>On the day of the experiment, count the cells and determine cell death by Trypan blue exclusion. Cell death should be less than 5%. Wash cells 1X in cold APBS, and centrifuge at 1,000 rpm at 4&deg;C for 10 min.</li>
 <li>Resuspend the cells in tumor culture medium at a density of 5 x 105 15/0 cells per 300 &mu;L.</li>
 <li>Transplant 5 x 105 cells in 300 &mu;L volume per frog by subcutaneous injection using a 25 gauge 5/8 needle on one side of the dorsal (back) side of the animal.</li>
 <li>Tumor growth will start within 2-3 weeks after tumor challenge. The initial tumor appearance must be noted and the tumor volume needs to be recorded every 2-3 days. Tumor volume (height X length X width) is measured using calipers.</li>
 <li>Once the tumor grows to 3,500 mm3 or the frog starts looking lethargic, it needs to be euthanized to prevent discomfort.</li>
</ol>
9. Reagents Needed:
<ul>
 <li>Amphibian Phosphate Buffered Saline (APBS): 6.6 g/L NaCl, 1.15 g/L Na2HPO4, 0.2 g/L KH2PO. pH to 7.5 using 10N NaOH and filter sterilized through 0.2 &mu;m filter.</li>
 <li>Tricaine Methane Sulfonate (TMS, MS-222) (Crescent Research Chemicals CAS#886-86-2).</li>
 <li>Sodium bicarbonate (Fisher Scientific S-233-500).</li>
 <li>Histopaque-1077 (Sigma-Aldrich 10771-100 mL)</li>
 <li>Heparin Sodium Salt (Sigma-Aldrich H3149-50KU)</li>
 <li>Culture medium for Xenopus 15/0 tumors [see 3 for more details]: 1 L of Iscove DMEM basal medium (Gibco-Invitrogen 11965) with 10 mL Insulin, 10 mL non-essential amino acids, 10 mL penicillin-streptomycin; 10 &mu;g/ mL of Kanamycin; 3 mL of primatone (Sheffield Products Division), 1 mL of &beta;2-mercaptoethanol, and 3.02 g NaHCO3 in water (pH 7.0). This medium is diluted to amphibian osmolarity by adding 30% double distilled water, and supplemented with 5% fetal bovine serum, 20% superantant from a Xenopus kidney cell line A6, and 0.25% of normal Xenopus serum.</li>
</ul>
10. Representative Results
<img src="/files/ftp_upload/2026/2026fig1.jpg" alt="Figure 1" /> 
 Figure 1. Stereomicroscopic analysis of skin graft rejection 12 days post-transplantation. LG-6 cloned frog received a skin graft from either (A) a MHC-identical LG-6 (shows no rejection) or (B) a MHC-disparate outbred donor (80% rejection). Arrows shows silvery iridophore pigmented cells marking healthy (non-rejected) grafted tissue. (*) Mark of forceps not due to rejection.
<img src="/files/ftp_upload/2026/2026fig2.jpg" alt="Figure 2" /> 
 Figure 2. Gp96 facilitates cross-presentation of tumor antigens in Xenopus. LG-15 PLs (5 x 105) were pulsed for 1 hr on ice either with APBS (negative control), 1 &mu;g of recombinant gp96 purified from an E. Coli culture, or 1 or 0.5 &mu;g of gp96 purified from 15/0 tumor tissue. After 3 washes, cells were adoptively transferred into LG-15 adult recipients (1 x 106/individual). Three days later, live 15/0 tumor cells (5 x 105) were transplanted by s.c. injection. Each curve represents the kinetics of tumor growth in one frog. Days post challenge when tumors first appeared were monitored, and tumor size was determined periodically with a caliper (length x width x thickness).

<ol>
	<li>Kobel, H. R., Pasquier, D. u., L, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperdiploid+species+hybrids+for+gene+mapping+in+Xenopus.">Hyperdiploid species hybrids for gene mapping in Xenopus.</a> Nature. 279, 157-158 (1979).</li><li>Robert, J., Menoret, A., Basu, S., Cohen, N., Srivastava, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+conservation+of+the+molecular+and+immunological+properties+of+the+chaperones+gp96+and+hsp70.">Phylogenetic conservation of the molecular and immunological properties of the chaperones gp96 and hsp70.</a> Eur J Immunol. 31, 186-195 (2001).</li><li>Robert, J., Gantress, J., Cohen, N., Maniero, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+as+an+experimental+model+for+studying+evolution+of+hsp--immune+system+interactions.">Xenopus as an experimental model for studying evolution of hsp--immune system interactions.</a> Methods. 32, 42-53 (2004).</li><li>Chardonnens, X., Pasquier, D. u., L, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+skin+allograft+tolerance+during+metamorphosis+of+the+toad+Xenopus+laevis:+a+possible+model+for+studying+generation+of+self+tolerance+to+histocompatibility+antigens.">Induction of skin allograft tolerance during metamorphosis of the toad Xenopus laevis: a possible model for studying generation of self tolerance to histocompatibility antigens.</a> Eur J Immunol. 3, 569-573 (1973).</li><li>Du Pasquier, L., Bernard, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+suppression+of+the+allogeneic+histocompatibility+reactions+during+the+metamorphosis+of+the+clawed+toad+Xenopus.">Active suppression of the allogeneic histocompatibility reactions during the metamorphosis of the clawed toad Xenopus.</a> Differentiation. 16, 1-7 (1980).</li><li>Ramanayake, T., Simon, D. A., Frelinger, J. G., Lord, E. M., Robert, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+study+of+T-cell+responses+to+skin+alloantigens+in+Xenopus+using+a+novel+whole-mount+immunohistology+method.">In vivo study of T-cell responses to skin alloantigens in Xenopus using a novel whole-mount immunohistology method.</a> Transplantation. 83, 159-166 (2007).</li><li>Robert, J., Ramanayake, T., Maniero, G. D., Morales, H., Chida, A. S., S, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+conservation+of+glycoprotein+96+ability+to+interact+with+CD91+and+facilitate+antigen+cross-presentation.">Phylogenetic conservation of glycoprotein 96 ability to interact with CD91 and facilitate antigen cross-presentation.</a> J Immunol. 180, 3176-3182 (2008).</li><li>Robert, J., Gantress, J., Rau, L., Bell, A., Cohen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minor+histocompatibility+antigen-specific+MHC-restricted+CD8+T+cell+responses+elicited+by+heat+shock+proteins.">Minor histocompatibility antigen-specific MHC-restricted CD8 T cell responses elicited by heat shock proteins.</a> J Immunol. 168, 1697-1703 (2002).</li><li>Morales, H., Robert, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+and+in+vitro+techniques+for+comparative+study+of+antiviral+T-cell+responses+in+the+amphibian+Xenopus.">In vivo and in vitro techniques for comparative study of antiviral T-cell responses in the amphibian Xenopus.</a> Biol Proced Online. 10, 1-8 (2008).</li><li>Maniero, G. D., Robert, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+conservation+of+gp96-mediated+antigen-specific+cellular+immunity:+new+evidence+from+adoptive+cell+transfer+in+xenopus.">Phylogenetic conservation of gp96-mediated antigen-specific cellular immunity: new evidence from adoptive cell transfer in xenopus.</a> Transplantation. 78, 1415-1421 (2004).</li></ol>

No conflicts of interest declared.

The amphibian Xenopus is a unique versatile non-mammalian model to study immunity. Its extensive use in biomedical and immunological research has yielded in many important research tools such as the MHC defined clones LG-6 and LG-15 as well as different cell lines and monoclonal antibodies. Using these tools we have established different in vitro and in vivo assays to study the ability of heat shock proteins such as gp96 to mediate potent Ag-specific anti-minor H-Ag and anti-tumor T cell respo...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Reagents needed:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Amphibian Phosphate Buffered Saline (APBS): </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NaCl, 1.15 g/L </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Na2HPO4, 0.2 g/L </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>KH2PO </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10N NaOH </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tricaine Methane Sulfonate (TMS, MS-222) </td>
<td>&nbsp;</td>
<td>Crescent Research Chemicals </td>
<td>CAS#886-86-2</td>
<td>&nbsp;</td>
<tr>
<td>Sodium bicarbonate </td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>S-233-500</td>
<td>&nbsp;</td>
<tr>
<td>Histopaque-1077</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>10771</td>
<td>100ml</td>
</tr>
<tr>
<td>Heparin Sodium Salt </td>
<td>&nbsp;</td>
<td>Sigma-Aldrich </td>
<td>H3149-50KU</td>
<td>&nbsp;</td>
<tr>
<td>Culture medium for Xenopus 15/0 tumors [see 3 for more details]:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Iscove DMEM basal medium</td>
<td>&nbsp;</td>
<td>Gibco-Invitrogen </td>
<td>11965</td>
<td>&nbsp;</td>
<tr>
<td>Insulin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Non-essential amino acids</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Kanamycin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Primatone</td>
<td>&nbsp;</td>
<td>Sheffield Products Division</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>&beta;2-mercaptoethanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NaHCO3 </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>30% double distilled water </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>5% featal bovine serum</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>20% superantant from a Xenopus kidney cell line A6 </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>0.25% of normal Xenopus serum</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Materials and Equipment:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50 and 15 ml conical centrifuge tubes (sterile)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>25 gauge 5/8 Precision Glide sterile needles</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>18 gauge 1&frac12; Precision Glide sterile needles</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1 ml Tuberculin Slip Tip Syringe sterile</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10 ml Slip Tip Syringe sterile</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1.5 ml MicroCentrifuge tubes (sterile)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>60 X 15 mm Polystyrene Petri Dishes sterile</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Razor blades</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>25 X 75 mm X 1 mm Premium Microscope Slides </td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10 cm glass petri dishes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>9" Pasteur Pipetes Durex Borosilicate Glass Cotton Plugged Disposable</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tygon tubing</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Two # 5 Swiss Jeweler&#x2019;s Forceps</td>
<td>&nbsp;</td>
<td>Miltex Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micro Dissecting Spring Scissors McPherson-Vannas straight cutting edge 6 mm</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Helios calipers </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>High intensity illuminator</td>
<td>&nbsp;</td>
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<td>Hemacytometer</td>
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<td>Un-bunsen burner</td>
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<td>37&deg;C shaking incubator</td>
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<td>Centrifuge</td>
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comparative in vivo study of gp96 adjuvanticity in the frog xenopus laevis

We have developed in the amphibian Xenopus laevis a unique non-mammalian model to study the ability of certain heat shock proteins (hsps) such as gp96 to facilitate cross-presentation of chaperoned antigens and elicit innate and adaptive T cell responses. Xenopus skin graft rejection provides an excellent platform to study the ability of gp96 to elicit classical MHC class Ia (class Ia) restricted T cell responses. Additionally, the Xenopus model system also provides an attractive alternative to mice for exploring the ability of gp96 to generate responses against tumors that have down-regulated their class Ia molecules thereby escaping immune surveillance. Recently, we have developed an adoptive cell transfer assay in Xenopus clones using peritoneal leukocytes as antigen presenting cells (APCs), and shown that gp96 can prime CD8 T cell responses in vivo against minor histocompatibility skin antigens as well as against the Xenopus thymic tumor 15/0 that does not express class Ia molecules. We describe here the methodology involved to perform these assays including the elicitation, pulsing and adoptive transfer of peritoneal leukocytes, as well as the skin graft and tumor transplantation assays. Additionally we are also describing the harvesting and separation of peripheral blood leukocytes used for flow cytometry and proliferation assays which allow for further characterization of the effector populations involved in skin rejection and anti-tumor responses.

Watch this Scientific Journal Video about Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis at JoVE.com

Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis

The frog Xenopus laevis provides an attractive alternative non-mammalian model for exploring the ability of heat shock protein such as gp96 to promote antigen-specific CD8 T cell responses. We present methods to study in vivo facilitation of cross-presentation of skin and tumor antigens by gp96.

Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis

We have developed in the amphibian Xenopus laevis a unique non-mammalian model to study the ability of certain heat shock proteins (hsps) such as gp96 to ...

comparative-vivo-study-gp96-adjuvanticity-frog-xenopus

Research

JoVE Journal

Immunology and Infection

10.5K Views.  University of Rochester. The frog Xenopus laevis provides an attractive alternative non-mammalian model for exploring the ability of heat shock protein such as gp96 to promote antigen-specific CD8 T cell responses. We present methods to study in vivo facilitation of cross-presentation of skin and tumor antigens by gp96.

Video: Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

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 ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...