Single cell analysis of multiple intracellular processes: ICAM-2/LFA-1 interactions as functional adhesion molecules of the immunological synapse

The molecular details of the intracellular signaling pathways for activation and differentiation of primary T cell interactions, a necessary component of adaptive immunity, have been difficult to ascertain. Mechanistic dissection of these processes, which often go awry in a number of immuno-pathophysiologies such as those involved in autoimmunity, requires the investigation of the molecules involved in the contact dependent interaction between T cells and antigen presenting cells.; Methodologies have been developed to identify T cell signaling molecules and determine functional characterization of the identified molecules. These pursuits have influenced the development of various technologies to study discrete biochemical events, such as kinase activities, phospholipids content, and nuclear translocation at the single cell level—allowing for the first time analysis of specific human lymphocyte subsets that exist within complex heterogeneous populations. These methodologies are needed to reveal the biology that is masked in conventional biochemical techniques. Multiplexing technologies have also been developed to simultaneously monitor production of various cytokines to obtain effector cell information and assess the state of T cell differentiation. Provision of these agents as intracellular determinants of biochemical events allow for a global assessment of intracellular signaling pathways at the single cell level of human primary cells. The usage of single cell techniques to characterize signaling events of multiple processes provides two major advantages (1) the ability to perform high throughput multiparametric experiments to identify the distinct signaling junctures of particular molecules of interest, (2) obtain a global understanding of immune cell processes, correlating parameters such as T cell activation, cytokine expression, and signaling intermediaries, simultaneously, at the single cell level. Mechanistic understanding of these pivotal processes will allow development of specific immunomodulatory agents, as well as provide a fundamental understanding of the combination of signals that dictate effector T cell differentiation—exposing this overlooked area for pharmacological intervention.