Genetically encoded reporter constructs that yield fluorescently labeled fusion proteins are a powerful tool for observing cell biological phenomena, but they have limitations. Sortagging (sortase-mediated transpeptidation) is a versatile chemoenzymatic system for site-specific labeling of proteins with small (<2 kDa) probes. Sortagging combines the precision of a genetically encoded tag with the specificity of an enzymatic reaction and the ease and chemical versatility of peptide synthesis. Here we apply this technique to proteins in vitro and on the surface of living cells.

Monoclonal antibodies that recognize specific antigens of interest are used as therapeutic agents and as tools for biomedical research. Discovering a single monoclonal antibody requires retrieval of an individual hybridoma from polyclonal mixtures of cells producing antibodies with a variety of specificities. The time required to isolate hybridomas by a limiting serial-dilution, however, has restricted the diversity and breadth of available antibodies. Here we present a soft lithographic method based on intaglio printing to generate microarrays comprising the secreted products of single cells. These engraved arrays enable a rapid (<12 h) and high-throughput (>100,000 individual cells) system for identification, recovery and clonal expansion of cells producing antigen-specific antibodies. This method can be adapted, in principle, to detect any secreted product in a multiplexed manner.

SUMMARY It has long been thought that clonal deletion efficiently removes almost all self-specific T cells from the peripheral repertoire. But here we found that self peptide-MHC specific CD8+ T cells in the blood of healthy humans were present in frequencies similar to those specific for non-self antigens. For the Y chromosome encoded SMCY antigen, self-specific T cells exhibited only a three-fold lower average frequency in males versus females and were anergic with respect to peptide activation, although this inhibition could be overcome by a stronger stimulus. We conclude that clonal deletion prunes but does not eliminate self-specific T cells and suggest that to do so would create holes in the repertoire that pathogens could readily exploit. In support of this hypothesis, we detected T cells specific for all 20 amino acid variants at the p5 position of a hepatitis C virus epitope in a random group of blood donors.

Rashidian et al. show that 89Zr-PEGylated single-domain antibodies that target CD8+ T cells can be used to monitor and evaluate the response to immunotherapy as a predictive tool.