Application of in vivo microscopy: evaluating the immune response in living animals

Until recently, the only method of demonstrating antigen processing and peptide–major histocompatibility complex (pMHC) formation by antigen-presenting cells (APCs) in vivo was to measure antigen-specific T cell activation in vitro [1,2]. Although these T cell-based assay systems are very sensitive, their drawbacks are variations in the stimulatory capacity of different APC populations and the unknown activation state of the responder T cells.Flow cytometry and tissue section imaging have been valuable methods for the investigation of antigen presentation in vivo. In particular, the use of pMHC-specific antibodies allows the detection of small numbers of molecules per cell, thereby permitting the analysis of antigen-specific T cell activation [3-5].The ability of a cell to move on any substrate must represent a combination between adhesion and the ability to extend processes. However, this obviously depends strongly on the nature of the surface; results on lymphocyte motility and interactions with APCs obtained from studies in vitro have consequently given drastically different results depending on the experimental system used [6-8]. In contrast, studies in vitro have provided valuable information about the signaling cascade that leads to lymphocyte activation, thereby describing the intricate choreography of key signaling molecules that participate in the formation of the immunological synapse at the T cell–APC interface [9,10]. Nevertheless, chemokine gradients, signals from the local nervous system and circulating hormones as well as integrin interactions with components of the extracellular matrix are lacking in cell culture systems. Finally, this methodology does not allow the observation of the movement and interaction of APCs with lymphocytes within organized lymphoid tissues in real time over short intervals.This has led several laboratories to develop imaging methods with high resolution to be able to perform spatiotemporal analysis of cell–cell interaction