ELISPOT assays provide reproducible results among different laboratories for T-cell immune monitoring--even in hands of ELISPOT-inexperienced investigators.

Measurements of antibodies in bodily fluids (e.g., by ELISA) have provided robust and reproducible results for decades and such assays have been validated for monitoring of B-cell immunity. In contrast, measuring T-cell immunity has proven to be a challenge due to the need to test live cells in functional assays ex vivo. Several previous efforts looking into the reproducibility of ex vivo T-cell assays between different laboratories, or even within the same laboratory, have provided rather discouraging results. The hypothesis we tested in this study is that those poor results are due to the lack of assay and data analysis standardization, rather than the inherent complexity of T-cell assays. In this study, 11 laboratories across Europe and the United States were provided identical reagents and were asked to follow the same protocol while testing aliquots of the same three cryopreserved peripheral blood mononuclear cells (PBMC) in an interferon-gamma (IFNgamma) ELISPOT assay measuring the antigen-specific T-cell response to a CMV peptide. All individuals performing the assays were ELISPOT novices. At their first attempt, while three of these individuals failed with the basic logistics of the trial, eight detected the peptide-specific CD8+ T-cells in frequencies approximating the values established by the Reference Laboratory. The data show that ELISPOT assays provide reproducible results among different laboratories when the assay procedure and data analysis is standardized. Since ELISPOT assays have been qualified and validated for regulated studies, they are ideal candidates for robust and reproducible monitoring of T-cell activity in vivo.

Synthetic and natural non-live vectors: rationale for their clinical development in cancer vaccine protocols.

Different arguments suggest that cytotoxic CD8 T lymphocytes (CTL) play a key role in the protection against tumors and in the establishment of anti-tumor immunity. Unfortunately, administration of soluble proteins alone generally does not induce CD8+ T cells presumably because antigen derived peptides are not introduced into the major histocompatibility complex (MHC) class I antigen presentation pathway. Attenuated recombinant live vectors such as viruses or bacteria which have the ability to deliver antigen into the cytosol of cells have been shown to induce cytotoxic T cell response. However, there are safety concerns associated with these approaches especially in immunodeficient patients. Synthetic vectors such as heat shock proteins, virus like particles (VLP) and liposomes could deliver exogenous protein into the cytosol of cells associated with the induction of CTL and tumor immunity. We and other groups have successfully exploited the original intracellular traffic of toxins to use them as vectors for tumor antigens.

Development of non-live vectors and procedures (liposomes, pseudo-viral particles, toxin, beads, adjuvantsellipsis) as tools for cancer vaccines.

Recombinant virus encoding tumor antigens are the most used vectors in human clinical trials of cancer vaccines because of their ability to target exogenous antigen in the endogenous MHC class I pathway and to elicit CTL. However, their use requires different constraining procedures to avoid their spreading. The immunosuppression of cancer patients may also increase their intrinsic toxicity. Therefore, the development of non-live vectors may avoid these drawbacks. Different groups now clearly demonstrated that particulate antigens when they are phagocytosed could be targeted in the MHC class I pathway. They also induce CTL in mice which when immunized with these particulate antigens were protected against a challenge with tumors expressing this antigen. Other strategies using toxins or antigens fused or incorporated into various oil or lipid based chemical adjuvants have also succeeded in the induction of CTL response and in some cases have been shown to be efficient as cancer vaccine.

The B subunit of Shiga toxin fused to a tumor antigen elicits CTL and targets dendritic cells to allow MHC class I-restricted presentation of peptides derived from exogenous antigens.

Immunization with peptide or recombinant proteins generally fails to elicit CTL, which are thought to play a key role in the control of virus-infected cells and tumor growth. In this study we show that the nontoxic B subunit of Shiga toxin fused to a tumor peptide derived from the mouse mastocytoma P815 can induce specific CTL in mice without the use of adjuvant. The Shiga B subunit acts as a vector rather than as an adjuvant, because coinjection of the tumor peptide and the B subunit as separate entities does not lead to CTL induction. We also demonstrated that in vitro the B subunit mediates the delivery of various exogenous CD8 T cell epitopes into the conventional MHC class I-restricted pathway, as this process is inhibited by brefeldin A and lactacystin and requires a functional TAP system. In contrast to other nonviral methods for transport of exogenous Ags into the endogenous MHC class I pathway that involve macropinocytosis or phagocytosis, the Shiga B subunit targets this pathway in a receptor-dependent manner, namely via binding to the glycolipid Gb3. Because this receptor is highly expressed on various dendritic cells, it should allow preferential targeting of the Shiga B subunit to these professional APCs. Therefore, the Shiga B subunit appears to represent an attractive vector for vaccine development due to its ability to target dendritic cells and to induce specific CTL without the need for adjuvant.

Cytokines and soluble cytokine receptor induction after IL-12 administration in cancer patients.

This study shows that subcutaneous administration of increasing doses of IL-12, once a week, in 21 cancer patients increased the expression of cytokine genes (interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), IP-10, MIG, IL-10, IL-4) in peripheral blood mononuclear cells even at very low doses (30 ng/kg). Surprisingly, no circulating TNF-alpha or IL-4 could be detected in the plasma of patients treated with IL-12. However, a marked increase of soluble IL-4 receptor was demonstrated in the plasma of five of the six patients studied, which may represent an additional mechanism by which IL-12 inhibits the development of the Th2 response in vivo. A marked decline of IFN-gamma and IP10 induction was recorded after repeated cycles of IL-12. In contrast, in most patients IL-12 increased IL-10 expression with no subsequent decrease during the course of therapy, and even an earlier peak of IL-10 induction at the 6th cycle. In addition, a constant up-regulation of serum soluble IFN-gamma receptor levels was observed after each cycle of IL-12 treatment with a delayed peak compared with the IFN-gamma peak. The constant rise of IL-10 and soluble IFN-gamma receptor during IL-12 therapy may therefore contribute to the inhibition of IFN-gamma activity detected after repeated cycles of IL-12. Lastly, a marked heterogeneity of cytokine induction was observed from one patient to another, which appeared to be independent of the dose of IL-12 administered. These data may lead to a better understanding of the biological activity of IL-12 and the in vivo mechanisms of its regulation.