Biophysics of immune cell signaling

Main text

Immune cell signaling is initiated at complex intercellular junctions or through multivalent interactions with ligands. Sites of immune receptor triggering are regulated by lipid composition, protein distributions, membrane geometry, and local forces. Downstream of receptor activation, mechanisms of signal propagation and regulation span from the molecular scale up to the tissue level and from milliseconds to hours in time. Cells can be potent as individuals and can also mobilize collective responses to an insult or injury. Fluorescence imaging has played a central role in exploring the interactions, organization, and dynamics that govern the evolution of immune cell interfaces and signaling and trafficking outcomes. Techniques ranging from single molecule to single cell are enabling insights that deepen our understanding of mechanosensing, ion channels, molecular arrangements at immune junctions, and more. The collection of papers in this special issue of Biophysical Journal help to advance our biophysical understanding of the processes that regulate innate and adaptive immune cell signaling.

A central theme in immune cell signaling is the formation and maintenance of the immunological synapse (IS). Despite decades of characterization of IS structure and behaviors, work in this issue from the Dustin lab has shown that there is still more to learn about this classic hallmark of T cell function. Capera et al. (1) quantified the spatiotemporal dynamics of Kv1.3, the primary voltage-gated potassium channel in human T cells. Kv1.3 activity sustains calcium signaling necessary for early T cell activation. At early times, they found Kv1.3 in the distal supramolecular activation clusters, colocalized with CD58/CD2. As the synapse matures, the channel is localized to the central supramolecular activation cluster, where it undergoes endocytosis that may act to terminate Kv1.3 signaling and limit T cell activation.

Adaptive immune responses rely on T cell and B cell interactions with antigen-presenting cells. Iliopoulou et al. (2) focused their study on a unique antigen-presenting cell type, subcapsular sinus macrophages (SSMs). These CD169⁺ macrophages line the subcapsular sinus floor and span the lymphatic endothelial layer to capture antigen. Antigen is transferred to the B cell follicle via long filopodia. The group isolated SSMs for in vitro studies by using multiplexed fluorescence microscopy and single-particle tracking. They found that SSMs under high extracellular matrix stiffness reduced both the length of filopodia and immune complex mobility. Understanding how the mechanical properties of the lymph node regulate SSM function may provide new avenues to modulate antigen delivery to B cells in vivo.

A growing field of immunotherapy is macrophage-mediated antibody-dependent cellular phagocytosis (ADCP). Jo et al. (3) examined the organization of immunoglobulin G-Fcγ receptor complexes at the macrophage/target-cell surface by using light sheet microscopy to image cell-cell interactions. They found that the mobility of the immunoglobulin G-engaged antigen on the target cell can influence the rearrangement of the Fcγ receptor on the macrophages, where higher mobility correlated with increased Syk phosphorylation. The authors then showed that the enhanced Syk activity drives Arp2/3 activity and actin polymerization, ultimately resulting in improved ADCP. This work adds the surface mobility of phagocytic ligands as a modulator of ADCP efficiency and a potential therapeutic target.

Soluble mediators, such as cytokines, also direct immune cell responses. Mazalo et al. (4) used microfluidics and lattice light sheet imaging to characterize the lateral distribution of chemokine receptors in T cells in the context of chemotactic repolarization. They found that the CCR5 receptors are uniformly distributed in migrating T cells, allowing for isotropic chemokine detection. In contrast, upon exposure to a CCL3 gradient, CCR5 is transiently localized to the uropod. They postulate that this redistribution of chemokine receptors may generate sensitivity to complex chemotactic inputs. Saed et al. (5) used single-particle tracking and super-resolution imaging to understand the spatiotemporal regulation of interleukin 2 (IL-2) during T cell activation. Typically, cytokine profiles are monitored by ensemble assays that cannot identify subcellular changes in distribution or dynamics. Here, Saed et al. were able to track IL-2-containing vesicles and found that their dynamics increased at 8 h after T cell receptor-mediated activation, the time frame at which peak IL-2 secretion is observed.

In addition to protein activities at the surface, composition of immune cell membranes may be highly consequential. van Deventer et al. (6) sought to determine how tetraspanins influence B cell function. Tetraspanins are small, four-transmembrane-domain scaffolds that contribute to the lateral organization of membrane proteins. van Deventer et al. combined flow cytometry, proximity ligation assays, and super-resolution to study CD37 and CD53, two tetraspanins found specifically on immune cells. They found that glycosylation is critical for the surface expression of CD37 but not CD53. Super-resolution imaging showed that the nanoscale organization of CD53 was also not dependent on glycosylation. However, CD53 interaction with CD45 and CD20 was glycosylation dependent, with glycosylation found to inhibit these interactions. The study by Yang et al. (7) used imaging fluorescence correlation spectroscopy and methyl-α-cyclodextrin-catalyzed lipid exchange to understand how lipids directly influence immune signaling. This work was performed in mast cells to understand how the lipids might contribute to recruitment of the Src family kinase Lyn, the initiating step of FcεRI signaling in response to cross-linking by multivalent allergen. They showed biophysical coupling of the membrane leaflets by substitutions of outer leaflet lipids that influenced inner leaflet mobility. Furthermore, they showed that by altering the inner leaflet to a more ordered state, both Syk recruitment and release of secretory contents increased, consistent with enhanced Lyn activity in the more ordered membrane. Together, these two studies add new information about the role of membrane organization in immune signaling regulation.

This special issue further includes four reviews that examine the role of mechanical forces in immune signaling, the organization of the chimeric antigen receptor-T cell synapse, and the engineering of defined antigens. Sengupta, Dillard, and Limozin review the current understanding of mechanics in T cell spreading, the first step in target recognition and engagement (8). They emphasize observations made by using different in vitro reconstitution strategies, coupled to imaging, with mobile or immobile ligands. They unify insights gained from these efforts into a morphodynamic model of immune cell scanning and activation that may be applicable to other immune cells. There is great interest in the comparisons between immune cell types to clarify their common and unique functions in homeostasis and disease responses. An important contribution to this debate comes from the groups of Salaita and Spillane in a review in which they directly compare the role of mechanical forces in regulating T cell receptor and B cell receptor activity (9). They discuss the triggering of the receptors and the forces at T cell and B cell synapses that contribute to their effector functions. The authors highlight how a biophysical understanding of the mechanical regulation of T and B cells can inform engineering of immune responses. The Su lab contributes a review of chimeric antigen receptor T cells and compares these therapeutic immune cells to their native T cell counterparts (10). Many biophysical aspects of chimeric antigen receptor-T cell-target junctions diverge from the physiological IS, perhaps providing the basis for differential sensitivity and signaling outcomes. Finally, a review from Hou and Treanor highlights the frontier technology of DNA origami that provides a programmable scaffold to control ligand geometries with nanometric precision and measure their impacts on immune cell fates (11). They discuss how different configurations of antigen presentation provide insights into T cell and B cell signaling thresholds.

This special issue contains outstanding examples of how biophysical research is revealing new mechanistic insights into immune cell signaling. Biophysical studies of immune cells have often been broadly applicable and paradigm shifting, for example, bringing forth the concepts of protein condensation in T cell signaling and lipid organization in B and T cells (12,13,14,15) that are now finding applications in many other systems. With new advances on the horizon, such as synthetic antibodies/antigens and the coupling of high-resolution structure to protein dynamics, biophysical approaches will continue to bring new and exciting perspectives to immune cell function.

Editor: Vasanthi Jayaraman.