The NF-cB-pSO polypeptide, a member of the Rel family of transcription factors, was produced as a fusion protein containing amino-terminal peptide additions that facilitate purification and detection with a monoclonal antibody and specific radiolabeling by phosphorylation in vitro. The 32P-labeled NF-#cB-p5O fusion polypeptide was used as the probe in Western blotting experiments and in screenings of a bacteriophage expression library to isolate cDNAs encoding interacting protein domains. As expected, cDNAs encoding proteins of the Rel family were identified. Surprisingly, the 32P-labeled NF-uB protein also specifically bound to proteins encoded by cDNAs for the human NF-1L6 transcription factor.The NF-#cB-p5O and NF-IL6 proteins directly interact, and the Rel homology domain and leucine-zipper motif, respectively, are important for this interaction. Since induction ofthe NF-ucB and NF-EL6 factors are important events in immune and acute-phase responses, this interaction could permit coregulation of genes.

Purpose Invariant NKT cells (iNKT) are innate-like CD1d-restricted T cells with immunoregulatory activity in diseases including cancer. iNKT from advanced cancer patients can have reversible defects including IFN-gamma production, and iNKT IFN-gamma production may stratify for survival. Previous clinical trials using iNKT cell activating ligand alpha-galactosylceramide have shown responses. Therefore, a phase 1 clinical trial was performed of autologous in vitro expanded iNKT cells in stage IIIB-IV melanoma. Experimental Design Residual iNKT cells (<0.05% of patient PBMC) were purified from autologous leukapheresis product using an antibody against the iNKT cell receptor linked to magnetic microbeads. iNKT cells were then expanded with CD3 mAb and IL-2 in vitro to obtain up to ~109 cells. Results Expanded iNKT cells produced IFN-gamma, but limited or undetectable IL-4 or IL-10. Three iNKT infusions each were completed on 9 patients, and produced only grade 1–2 toxicities. The 4th patient onward received systemic GM-CSF with their second and third infusions. Increased numbers of iNKT cells were seen in PBMC after some infusions, particularly when GM-CSF was also given. IFN-gamma responses to alpha-galactosylceramide were increased in PBMC from some patients after infusions, and DTH responses to Candida increased in 5/8 evaluated patients. Three patients have died, three were progression-free at 53, 60 and 65 months, three received further treatment and were alive at 61, 81, and 85 months. There was no clear correlation between outcome and immune parameters. Conclusions Autologous in vitro expanded iNKT cells are a feasible and safe therapy, producing Th1-like responses with anti-tumor potential.

Lipids and glycolipid molecules derived from Mycobacterium tuberculosis can be presented to T cells by CD1 cell-surface molecules in humans. These lipid-specific T cells are cytolytic, secrete pro-inflammatory cytokines and have bactericidal activity. Here, we describe studies in which lipids from M. tuberculosis were incorporated into liposomes with adjuvant and tested as vaccines in a guinea pig aerosol tuberculosis challenge model. Animals vaccinated with mycobacterial lipids showed reduced bacterial burdens in the lung and spleen at 4 weeks after infection. In addition, the lungs of lipid-vaccinated animals also had significantly less pathology, with granulomatous lesions being smaller and more lymphocytic. In contrast, animals receiving only vehicle control immunizations had granulomatous lesions that were larger and often contained caseous necrotic centers. Quantification of histopathology by morphometric analysis revealed that the overall percentage of lung occupied by diseased tissue was significantly smaller in lipid-vaccinated animals as compared to vehicle control animals. In addition, the mean area of individual granulomatous lesions was found to be significantly smaller in both lipid- and bacillus Calmette-Guerin-vaccinated guinea pigs. These data support an important role for lipid antigens in the immune response to M. tuberculosis infection, potentially through the generation of CD1-restricted T cells. Immunogenic lipids thus represent a novel class of antigens that might be included to enhance the protective effects of subunit vaccine formulations.

The Ly‐6 alloantigens have been shown to play a critical role in T lymphocyte activation. To isolate a Ly‐6 cDNA, synthetic oligonucleotides, based on the partial amino acid sequence of purified Ly‐6E.1 protein, were used to probe a cDNA library. The synthetic oligonucleotides or the isolated cDNA detected a 1.1‐kb RNA species. Sequence analysis of the cDNA clone revealed that the Ly‐6E.1 protein consists of a 26‐amino acid leader followed by a 108‐residue, cysteine‐rich, core protein with no N‐linked glycosylation sites. Southern blot analysis of genomic DNAs revealed multiple bands indicating a family of related genes. Using recombinant inbred and Ly‐6 congenic strains of mice, restriction fragment length polymorphisms were demonstrable, and correlated with the Ly‐6 allotype of the DNA donors. This probe will enable further molecular genetic analysis of the role of Ly‐6‐linked proteins in the process of T lymphocyte activation. Isolation of Ly‐6 genomic clones may promote a further understanding of the complex tissue‐specific expression patterns characteristic of Ly‐6‐linked genes.