Advances in In Vivo Imaging Techniques for the Visualization and Quantification of DC-T Cell Interactions

Abstract

Short CommunicationSince the first application of two photon intravital microscopy (2PM) to the study of the immune system in 1997 [1], 2PM imaging has offered several major advantages over previous techniques for study immune interactions. Such as 2D plate based culture systems, as cells exhibit significantly different migratory behavior in 2D as compared to 3D. Whilst, 3D matrigel systems allow cells to migrate in a more natural manner, they fail to adequately replicate a physiological environment which is densely packed with other cell types and contains a myriad of environmental cues such as chemokines, adhesion molecules and lymphatic flow, these factors are largely overcome by the use of ex-vivo/explant organ imaging. However, this system also has significant limitations in that cells cannot be recruited to or exit the organ of interest and due to limitations in tissue oxygenation, imaging sessions can generally only be conducted for less than one hour [2]. Therefore, the use of intravital 2PM has opened up a new era of understanding in immunology. Especially in terms of how we view the importance of the migratory behavior and the interactions that occur between cells of the immune system under both homeostatic and inflammatory conditions. Perhaps this has been most enlightening when applied to the study of the adaptive immune system and the interaction between CD4+ and CD8+ T cells with dendritic cells (DC). These studies have revealed that T cells are constantly forming non-cognate interactions with DC as they migrate through the lymph node (LN) at an average velocity of 10μm/min, allowing individual DC to briefly interact with an estimated 5000 T cells an hour [3,4]. This behavior allows for the rapid scanning of the TCR repertoire during an immune response and the swift activation of antigen specific T-cells. Initial cognate interactions consist of brief phase I interactions (~0-6 h) typically lasting 10-30 minutes each, before progression to phase II interactions where stable immune synapses are formed at ~6-18 h post-activation, a transition to phase III interactions then occurs between ~18-30 h, which is typified by multiple short lived interactions and a pattern of swarming behavior [5,6]. The quality or quantity of the cognate antigen and both the phenotype and activation status of the stimulatory DC have all been shown to influence the process of activation and differentiation [7,8], and the application of novel techniques for probing this process will undoubtedly allow for a much greater understanding of the adaptive immune response. Tracking with Novel Fluorescent Proteins/Dyes:The capabilities and application of intravital 2PM for the analysis of cellular processes and the dynamics of interactions has seen an exponential growth with the introduction of several novel fluorescent proteins, optical markers and NIR (Near Infrared) fluorophores. The use of optical markers such as photoactivatable (PA) [9] fluorescent proteins PA-GFP and photo-convertible fluorophores (e.g. Kaede [10] and kikGR [1...