CD8+ T lymphocytes recognize antigens as short peptides bound to MHC class I molecules. Available methods cannot determine the number and distribution of these ligands on individual cells or detect antigen-presenting cells in tissues. Here we describe a method for eliciting and identifying monoclonal antibodies specific for a particular peptide-MHC class I combination. One such antibody can identify antigen complexes with a limit of detection approaching that of T cells. We used this antibody to determine the number of peptide-class I complexes generated upon viral infection, to identify antigen-presenting cells in cell mixtures, to determine the site of peptide-MHC class I interaction inside cells, and to visualize cells bearing specific peptide-MHC class I complexes after in vivo infection. Similar antibodies may prove useful for diagnostic or therapeutic purposes in cancer, infectious diseases, and autoimmune disorders.

Dendritic cells (DC) are potent antigen-presenting cells that initiate and regulate T-cell responses. In this study, the numbers and functional cytokine secretions of plasmacytoid and myeloid DC (pDC and mDC, respectively) in peripheral blood from young and elderly subjects were compared. Overall, pDC numbers in peripheral blood were lower in healthy elderly compared with healthy young subjects (p = 0.016). In response to influenza virus stimulation, isolated pDC from healthy elderly subjects secreted less interferon (IFN)–α compared with those from healthy young subjects. The decline in IFN-α secretion was associated with a reduced proportion of pDC that expressed Toll-like receptor–7 or Toll-like receptor-9. In contrast, there was little difference in the numbers and cytokine secretion function between healthy young and healthy elderly subjects (p = 0.82). However, in peripheral blood from frail elderly subjects, the numbers of mDC were severely depleted as compared with either healthy young or elderly subjects (p = 0.014 and 0.007, respectively). Thus, aging was associated with the numerical and functional decline in pDC, but not mDC, in healthy young versus elderly subject group comparisons, while declining health in the elderly can profoundly impact mDC negatively. Because of the importance of pDC for antiviral responses, the age-related changes in pDC likely contribute to the impaired immune response to viral infections in elderly persons, especially when combined with the mDC dysfunction occurring in those with compromised health.

The application of pulsed electric fields to cells is extended to include nonthermal pulses with shorter durations (10-300 ns), higher electric fields (< or =350 kV/cm), higher power (gigawatts), and distinct effects (nsPEF) compared to classical electroporation. Here we define effects and explore potential application for nsPEF in biology and medicine. As the pulse duration is decreased below the plasma membrane charging time constant, plasma membrane effects decrease and intracellular effects predominate. NsPEFs induced apoptosis and caspase activation that was calcium-dependent (Jurkat cells) and calcium-independent (HL-60 and Jurkat cells). In mouse B10-2 fibrosarcoma tumors, nsPEFs induced caspase activation and DNA fragmentation ex vivo, and reduced tumor size in vivo. With conditions below thresholds for classical electroporation and apoptosis, nsPEF induced calcium release from intracellular stores and subsequent calcium influx through store-operated channels in the plasma membrane that mimicked purinergic receptor-mediated calcium mobilization. When nsPEF were applied after classical electroporation pulses, GFP reporter gene expression was enhanced above that observed for classical electroporation. These findings indicate that nsPEF extend classical electroporation to include events that primarily affect intracellular structures and functions. Potential applications for nsPEF include inducing apoptosis in cells and tumors, probing signal transduction mechanisms that determine cell fate, and enhancing gene expression.

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