Characterizing porcine invariant natural killer T cells: A comparative study with NK cells and T cells.

CD1d-restricted invariant natural killer T (iNKT) cells are innate-like T cells that share phenotypic characteristics of both NK and conventional T cells (Tconv). Although iNKT cells have been well characterized in mice and humans, functional CD1d and CD1d-restricted iNKT cells are not universally expressed in mammals. Swine express iNKT cells that can be detected using α-galactosylceramide (α-GalCer)-loaded CD1d tetramers. In the present study, we characterized iNKT cells from the blood, spleen, lymph node, lung and liver of commercial mixed-breed pigs, and compared their phenotype to NK cells and Tconv. The principal findings are that pig iNKT cells are CD8α and CD44 positive and CD11b and Nkp46 negative. Most are also negative for the CD4 co-receptor, which is used to distinguish functionally distinct mouse and human iNKT cells subsets. The frequency of IFN-γ-producing CD8αbright iNKT cells was 3-4-fold higher than CD8αdull iNKT cells, suggesting that CD8α expression identifies iNKT cells with a unique functional role in immune responses. Finally, large variability was detected among pigs in interactions between iNKT cells and monocytes when iNKT cells were activated with α-GalCer, which raises a cautionary note about manipulating iNKT cells for immunotherapy. Collectively, our study provides important phenotypic and functional information about porcine iNKT cells that will be useful for understanding how iNKT cells contribute to immune responses in swine, with potential implications for human health.

Multi-peptide vaccines vialed as peptide mixtures can be stable reagents for use in peptide-based immune therapies.

To date, most peptide-based vaccines evaluated for the treatment of cancer have consisted of one or few peptides. However, as a greater number of peptide antigens become available for use in experimental therapies, it is important to establish the feasibility of combining multi-peptide reagents as individual peptide mixtures. We have found that mixtures of up to 12 peptides can be analyzed accurately for identity, purity, and stability (for at least 5 years) using a combination of high-performance liquid chromatography (HPLC) and mass spectrometry and these complex peptide mixtures have been acceptable for use in human clinical trials. We have also identified some specific concerns for degradation products that should be considered in multi-peptide vaccine preparation and follow-up quality assurance studies. Results from these analyses have implications for changing the way peptide-based vaccines are manufactured and demonstrate that multi-peptide vaccines are reliable reagents for use in peptide-based immune therapies.

Identification of class I MHC-associated phosphopeptides as targets for cancer immunotherapy.

Alterations in phosphorylation of cellular proteins are a hallmark of malignant transformation. Degradation of these phosphoproteins could generate cancer-specific class I MHC-associated phosphopeptides recognizable by CD8+ T lymphocytes. In a comparative analysis of phosphopeptides presented on the surface of melanoma, ovarian carcinoma, and B lymphoblastoid cells, we find 5 of 36 that are restricted to the solid tumors and common to both cancers. Differential presentation of these peptides can result from differential phosphorylation of the source proteins. Recognition of the peptides on cancer cells by phosphopeptide-specific CD8+ T lymphocytes validates the potential of these phosphopeptides as immunotherapeutic targets.