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

Summary

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.

Abstract

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.

Protocol

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).

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Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents needed:
Amphibian Phosphate Buffered Saline (APBS):
NaCl, 1.15 g/L
Na2HPO4, 0.2 g/L
KH2PO
10N NaOH
Tricaine Methane Sulfonate (TMS, MS-222)	Crescent Research Chemicals	CAS#886-86-2
Sodium bicarbonate	Fisher Scientific	S-233-500
Histopaque-1077	Sigma-Aldrich	10771	100ml
Heparin Sodium Salt	Sigma-Aldrich	H3149-50KU
Culture medium for Xenopus 15/0 tumors [see 3 for more details]:
Iscove DMEM basal medium	GIBCO, by Life Technologies	11965
Insulin
Non-essential amino acids
Penicillin-streptomycin
Kanamycin
Primatone	Sheffield Products Division
β2-mercapt–thanol
NaHCO3
30% double distilled water
5% featal bovine serum
20% superantant from a Xenopus kidney cell line A6
0.25% of normal Xenopus serum
Materials and Equipment:
50 and 15 ml conical centrifuge tubes (sterile)
25 gauge 5/8 Precision Glide sterile needles	BD Biosciences
18 gauge 1½ Precision Glide sterile needles	BD Biosciences
1 ml Tuberculin Slip Tip Syringe sterile	BD Biosciences
10 ml Slip Tip Syringe sterile	BD Biosciences
1.5 ml MicroCentrifuge tubes (sterile)
60 X 15 mm Polystyrene Petri Dishes sterile	Falcon BD
Razor blades
25 X 75 mm X 1 mm Premium Microscope Slides	Fisher Scientific
10 cm glass petri dishes
9" Pasteur Pipetes Durex Borosilicate Glass Cotton Plugged Disposable	VWR international
Tygon tubing
Two # 5 Swiss Jeweler’s Forceps	Miltex Inc.
Micro Dissecting Spring Scissors McPherson-Vannas straight cutting edge 6 mm	Roboz Surgical Instruments Co.
Helios calipers
Dissecting microscope
High intensity illuminator
Hemacytometer
Un-bunsen burner
37°C shaking incubator
Centrifuge