The cytosolic 70-kDa heat shock proteins, or HSP70, are evolutionarily ancient and highly conserved intracellular molecular chaperones that, like other heat shock proteins (HSPs), help with intracellular transport, folding of newly synthesized proteins, and prevention of protein aggregation (1
). Increasing evidence suggests that these proteins are critical players in immune surveillance and tumor immunity (2
). The ability of HSP70 to mediate potent anti-tumor responses is due to its intrinsic ability to activate both the adaptive and the innate arms of the immune system (2
). HSP70 can elicit antigen (Ag)-dependent responses, in which chaperoned antigenic peptides are cross-presented by antigen-presenting cells (APCs) through classical MHC class Ia (class Ia) molecules for CD8+ T cell recognition and induction of strong cytotoxic T lymphocyte (CTL) responses (5
). On the other hand, HSP70 also induces a strong peptide-independent pro-inflammatory response (2
). This is due to the interaction of HSP70 with a multitude of signaling receptors such as CD14, Toll-like receptor (TLR) 2, TLR 4, and CD40 that are involved in activation of the innate immune system (7
Due to these immunological properties, HSP70 is a prime candidate as an anti-tumor vaccine. However, the only HSP that has been evaluated to date in clinical trials is gp96 (10
), despite many unknowns associated with this protein. For instance, it is not known how peptide binding occurs in vivo
, and it is difficult to associate gp96 with peptides in vitro
). Conversely, HSP70 represents a potentially more reliable anti-tumor vaccine candidate, since it has defined peptide binding stoichiometry and requirements that are dependent on ATP to ADP hydrolysis (11
). Furthermore, anti-tumor immunity generated by tumor-derived HSP70-peptide complexes is evolutionarily conserved since it has been demonstrated in the amphibian Xenopus
There is, however, an unresolved issue: purification of HSP70 from tissues does not distinguish between the inducible hsp72 and the constitutive hsc73, which are the two different cytosolic members of the HSP70 family encoded by different genes with different regulation. Hsc73 is constitutively expressed and poorly stress-inducible, while hsp72 is rapidly induced under stress conditions including oxidative stress, changes in pH, as well as malignancy (1
). Therefore, one cannot clearly attribute immune protection to either protein. Interestingly, even though HSP70 (i.e., pool of cytosolic hsp72 and hsc73) is one of the prime candidates for use in tumor immunotherapy, there has been minimal attempt to evaluate the respective contribution of these two HSP70 forms in stimulating anti-tumor responses, especially in the case of tumors that downregulate class Ia expression to escape immune recognition. Some evidence suggests that hsp72 may be the key player in these responses. For example, in a rat carcinoma model, hsp72 expression correlated with tumor immunogenicity (17
). Also, hsp72 was found to bind more peptides, more efficiently under oxidative conditions than hsc73 (18
). It is noteworthy, however, that these data do not rule out the possible immunogenicity of hsc73.
Although the relationships between class Ia molecules and HSPs, including HSP70, have been studied extensively, there is little known regarding the roles of non-classical MHC class Ib (class Ib) gene products. Class Ib molecules are heterogeneous genes structurally similar to class Ia, but exhibit low polymorphism and limited tissue distribution. Interestingly, many tumors, especially malignant cancers, downregulate class Ia expression to avoid immune detection (19
). However, many of these tumors still express class Ib molecules (13
). Indeed, class Ib molecules are increasingly implicated in immune surveillance, especially in tumor metastasis where class Ia expression is lost (20
). On the other hand, there is strong evidence highlighting the pro-tumorigenic roles of certain class Ib molecules. For instance, patients with malignant breast tumors that have lost class Ia expression, but still express class Ib molecules such as HLA-E and HLA-G, result in more severe clinical outcomes, relapse, and poor prognosis (21
). Therefore, the roles of these class Ib molecules in cancer are still largely unclear and warrant further examination.
In the present work, we took advantage of our non-mammalian Xenopus
comparative tumor immunity model to test the difference in immunogenicity of hsp72 and hsc73 in class Ib-mediated anti-tumor responses. Xenopus
is an attractive animal model for studying HSP-mediated immune responses due to its poor responsiveness to lipopolysaccharide (LPS) (4
). For this purpose, we used the transplantable class Ia negative thymic tumor 15/0, which expresses several class Ib genes. Using this model, we were able to show that hsp72, but not hsc73, can prime class Ib-mediated anti-tumor responses in an Ag-dependent manner.