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 contribut...

<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>

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>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hemacytometer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Un-bunsen burner</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>37&deg;C shaking incubator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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

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JoVE Journal

Immunology and Infection

10.5K Views.  University of Rochester. La rana Xenopus laevis Offre una interessante alternativa non-mammiferi modello per esplorare la possibilità di proteine ​​da shock termico come gp96 per promuovere antigene-specifiche risposte delle cellule T CD8. Vi presentiamo i metodi per lo studio In vivo di cross-presentazione di antigeni tumorali della pelle e da gp96.