Emmanuel Katsanis

Because of significant medical advances in the past 50 years, the number of adult survivors of childhood/adolescent cancer has increased dramatically. Unfortunately, more than 60% of these survivors will have at least 1 long-term side effect from treatment. This growing population requires dedicated care by their primary physicians because they have specific risk factors depending on their initial cancer diagnosis and the treatment modalities they received. Internists and family physicians play an integral role in providing appropriate screening, treatment, and counseling to prevent morbidity and mortality in these patients.

T lymphocytes activated by dendritic cells (DC) which present tumor antigens play a key role in the antitumor immune response. However, in patients suffering from active cancer, DC are not efficient at initiating and supporting immune responses as they participate to T lymphocyte inhibition. DC in the tumor environment are functionally defective and exhibit a characteristic of immature phenotype, different to that of DC present in nonpathological conditions. The mechanistic bases underlying DC dysfunction in cancer responsible for the modulation of T-cell responses and tumor immune escape are still being investigated. Using two different mouse tumor models, we showed that tumor-infiltrating DC (TIDC) are constitutively immunosuppressive, exhibit a semimature phenotype, and impair responder T lymphocyte proliferation and activation by a mechanism involving CD39 ectoenzyme.

We have previously reported on the anti-tumoral potential of a chaperone-rich cell lysate (CRCL) vaccine. Immunization with CRCL generated from tumors elicits specific T and NK cell-dependent immune responses leading to protective immunity in numerous mouse tumor models. CRCL provides both a source of tumor antigens and danger signals leading to dendritic cell activation. In humans, tumor-derived CRCL induces dendritic cell activation and CRCL-loaded dendritic cells promote the generation of cytotoxic T lymphocytes in vitro. The current study was designed to identify the signaling events and modifications triggered by CRCL in antigen presenting cells. Our results indicate that tumor-derived CRCL not only promotes the activation of dendritic cells, but also significantly fosters the function of macrophages that thus appear as major targets of this vaccine. Activation of both cell types is associated with the induction of the MAP kinase pathway, the phosphorylation of STAT1, STAT5 and AKT and with transcription factor NF-kappaB activation in vitro and in vivo. These results thus provide important insights into the mechanisms by which CRCL-based vaccines exert their adjuvant effects on antigen presenting cells.

Although the use of tumor-derived heat shock/chaperone proteins (HSPs) as anticancer vaccines is gaining wider study and acceptance, there have thus far been no reports concerning chaperone antitumor activities against disseminated hematological malignancies. We have devised an efficient and effective method for purification of the chaperone proteins grp94/gp96, HSP90, HSP70, and calreticulin from harvested A20 murine leukemia/lymphoma tumor material. We have demonstrated that these purified proteins, when used as vaccines, can induce potent and specific immunity against a lethal tumor challenge. Individual chaperone proteins were differentially effective in their abilities to provide immune protection. The increase in survival generated by the most effective chaperone vaccine, HSP70, resulted from at least a 2-log reduction in tumor burden. Syngeneic granulocyte macrophage colony-stimulating factor producing fibroblasts were injected at the site of vaccination in an attempt to augment the immune response. Surprisingly, localized granulocyte macrophage colony-stimulating factor production inhibited the protective effects of chaperone vaccination. These studies provide evidence that chaperone proteins can be isolated from B-cell tumors and used effectively to immunize against disseminated lymphoid malignancies.

The endoplasmic reticulum (ER) is a major site of passage for proteins en route to other organelles, to the cell surface, and to the extracellular space. It is also the transport route for peptides generated in the cytosol by the proteasome into the ER for loading onto major histocompatibility complex class I (MHC I) molecules for eventual antigen presentation at the cell surface. Chaperones within the ER are critical for many of these processes; however, outside the ER certain of those chaperones may play important and direct roles in immune responses. In some cases, particular ER chaperones have been utilized as vaccines against tumors or infectious disease pathogens when purified from tumor tissue or recombinantly generated and loaded with antigen. In other cases, the cell surface location of ER chaperones has implications for immune responses as well as possible tumor resistance. We have produced heat-shock protein/chaperone protein-based cancer vaccines called "chaperone-rich cell lysate" (CRCL) that are conglomerates of chaperones enriched from solid tumors by an isoelectric focusing technique. These preparations have been effective against numerous murine tumors, as well as in a canine with an advanced lung carcinoma treated with autologous CRCL. We also published extensive proteomic analyses of CRCL prepared from human surgically resected tumor samples. Of note, these preparations contained at least 10 ER chaperones and a number of other residents, along with many other chaperones/heat-shock proteins. Gene ontology and network analyses utilizing these proteins essentially recapitulate the antigen presentation pathways and interconnections. In conjunction with our current knowledge of cell surface/extracellular ER chaperones, these data collectively suggest that a systems-level view may provide insight into the potent immune stimulatory activities of CRCL with an emphasis on the roles of ER components in those processes.