Comparative <em>in vivo</em> Study of gp96 Adjuvanticity in the Frog <em>Xenopus laevis</em>

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.

Inhibition of Local Immune Responses by the Frog-Killing Fungus Batrachochytrium dendrobatidis

“…The present study reproduces these in vitro observations in vivo using a modified protocol to induce inflammatory swelling in the feet of X. laevis. Primary injection with PHA or killed E. coli induces a primarily innate immune response mainly causing an infiltration of phagocytic leukocytes (19,23,27,29). The inflammatory swelling induced by these injections was not significantly affected when B. dendrobatidis supernatants were simultaneously injected, suggesting no impairment of innate immune responses.…”

“…We assumed that injection of E. coli induces a rapid immigration of neutrophils and macrophages rather than a lymphocyte- specific response because these cells are recruited during a single intraperitoneal injection of E. coli into X. laevis (19,23). However, we did not test whether intraperitoneal priming with E. coli would result in enhanced swelling of the foot, as was observed with priming and injection of PHA.…”

“…In our experimental design, we injected PHA intraperitoneally 7 days before the second injection of PHA into the feet. The peritoneum was chosen for PHA priming so both feet did not have to be injected and because the peritoneum has previously been used as the site to induce immune responses in X. laevis (18,19,23).…”

“…Phytohemagglutinin (PHA), phorbol-12-myristate 13-acetate, concanavalin A, and killed bacteria are mitogenic for amphibian B and T lymphocytes (19,21). Also, the intraperitoneal injection of killed Escherichia coli into X. laevis induces infiltration of macrophages and neutrophils into the peritoneum (19,23).…”

“…A primarily innate immune response was induced with killed E. coli, known to stimulate phagocyte infiltration into the peritoneum (19,23). Injection of PHA was chosen as a way to induce mixed innate and adaptive immune responses.…”