Photochemical approaches to T-cell activation

Abstract

Despite decades of intensive research, T-cell activation has remained mysterious because of both the dizzying diversity of antigen recognition and the speed and comprehensiveness of the T-cell-receptor signalling network. Further progress will require new approaches and reagents that provide added levels of control. Photochemistry allows specific biochemical processes to be controlled with light and is well suited to mechanistic studies in complex cellular environments. In recent years, several laboratories have adopted approaches based on photoreactive peptide-major histocompatibility complex reagents in order to study T-cell activation and function with high precision. Here, I review these efforts and outline future directions for this exciting area of research.

Figure 1

Photoactivatable and conditional peptide-major histocompatibility complex (pMHC). In photoactivatable pMHC (left), a photocleavable protecting group is placed on a peptide side chain that is required for high-affinity interaction with the T-cell receptor (TCR). UV irradiation cleaves off this group, yielding the agonist pMHC complex. In conditional pMHC (right), a photocleavable group is attached to a peptide bond within the peptide. UV irradiation cleaves the peptide in two, after which it dissociates from the MHC protein. Grey, MHC protein; green, peptide; cyan, photocaged side chain; magenta, photocleavable peptide bond.

Figure 2

A typical peptide-major histocompatibility complex (pMHC) photoactivation experiment. T cells bearing fluorescent signalling probes are attached to glass surfaces containing photoactivatable (photoact.) pMHC, and a portion of the surface is irradiated with focused ultraviolet (UV) light during an imaging experiment. Below, a time-lapse montage shows consecutive images of a single cell expressing signalling probes for diacylglycerol (DAG, green) and the microtubule-organizing centre (MTOC) (red). The time and region of UV irradiation is indicated by the purple and yellow circle. Accumulation of DAG at the irradiated region [imaged using total internal reflection fluorescence (TIRF) microscopy] precedes the re-orientation of the MTOC. Above, a schematic diagram is shown that corresponds generally to the images presented below. The UV irradiation event is indicated by a purple and yellow beam. The objective lens (Obj.) in the schematic indicates the perspective of the images below.

Figure 3

Applications of conditional peptide-major histocompatibility complex (pMHC) technology. Ultraviolet (UV) irradiation of conditional pMHC in the presence of rescue peptides leads to the formation of pMHC complexes containing the rescue peptides. This enables the rapid generation of pMHC tetramers that can be used for epitope discovery, immune monitoring, or T-cell selection. Grey, MHC protein; green and magenta, conditional ligand; rescue peptides are shown in different colours.