Cross Talk between CD3 and CD28 Is Spatially Modulated by Protein Lateral Mobility

Abstract

dFunctional convergence of CD28 costimulation and TCR signaling is critical to T-cell activation and adaptive immunity. These receptors form complex microscale patterns within the immune synapse, although the impact of this spatial organization on cell signaling remains unclear. We investigate this cross talk using micropatterned surfaces that present ligands to these membrane proteins in order to control the organization of signaling molecules within the cell-substrate interface. While primary human CD4 ؉ T cells were activated by features containing ligands to both CD3 and CD28, this functional convergence was curtailed on surfaces in which engagement of these two systems was separated by micrometer-scale distances. Moreover, phosphorylated Lck was concentrated to regions of CD3 engagement and exhibited a low diffusion rate, suggesting that costimulation is controlled by a balance between the transport of active Lck to CD28 and its deactivation. In support of this model, disruption of the actin cytoskeleton increased Lck mobility and allowed functional T-cell costimulation by spatially separated CD3 and CD28. In primary mouse CD4؉ T cells, a complementary system, reducing the membrane mobility increased the sensitivity to CD3-CD28 separation. These results demonstrate a subcellular reaction-diffusion system that allows cells to sense the microscale organization of the extracellular environment. Spatial organization plays important roles in cell signaling, governing a wide range of functions, including migration, polarization, and morphogenesis. A striking example at subcellular scales has emerged in the immune synapse (IS), a small (ϳ70-m 2 ) area of contact between a lymphocyte and an antigen-presenting cell (APC) which serves as a platform that focuses and modulates cell-cell communication. The archetypal IS formed between a T cell and an APC contains a central supramolecular activation cluster (cSMAC) of T-cell receptor (TCR)-pMHC complexes surrounded by a peripheral supramolecular activation cluster (pSMAC) with LFA-1-ICAM-1 (1-3). The interfaces of different T-cell-APC pairings exhibit variations on this "bullseye" pattern (4-10), and manipulation of IS structure modulates T-cell activation (11-13), suggesting that microscale organization contributes to the language of cell-cell communication. However, the concept that signaling can be modulated at such scales places stringent requirements on the dynamics of intracellular signaling molecules (14-17), and experimental examples of such mechanisms, particularly within the small dimensions of the IS, have been elusive.We focus here on spatially resolved, microscale cell signaling in the context of CD28 costimulation. When bound by CD80 or CD86, typically presented by an APC in conjunction with pMHC, CD28 augments TCR signaling and is essential for full activation of naive T cells. A role of spatial organization in this signaling was established by experiments in which CD28 was engaged outside the IS, a trans-costimulation configuration representing the action...

Live-Cell Super-resolution Reveals F-Actin and Plasma Membrane Dynamics at the T Cell Synapse

“…Studies have shown that the cortical actin meshwork and the plasma membrane may interact directly (14) or indirectly via transmembrane and scaffolding proteins (4,15,16). We therefore hypothesized that the flow of the cortical actin mesh might be coupled to the membrane, causing retrograde membrane flow.…”

“…The cortical actin meshwork and the plasma membrane may interact directly (14) or indirectly via transmembrane and scaffolding proteins (4,15,16) including TCR (17), which has been observed to cause modulation of protein lateral mobility including trapping, tethering, and corralling (18). These sites of interaction frequently occur at areas of high membrane lipid order (12,19,20).…”

T cell activation and immune synapse organization respond to the microscale mechanics of structured surfaces

“…Except for the experiment of Fig. 3D, IFN-γ secretion was measured using a surface capture assay (Miltenyi) as previously described (17,18,27). Fig.…”

Linking form to function: Biophysical aspects of artificial antigen presenting cell design

“…Similarly, mechanically trapping TCR nanoclusters at the peripheral regions of a forming synapse significantly prolonged and strengthened signaling from TCR nanoclusters [106]. However, separation of anti-CD3 and anti-CD28 contact sites by several microns in human T cells curtailed co-stimulatory activity [107]. Thus, while existing aAPC fabrication techniques generally rely on randomly distributed ligands, these findings suggest it may be possible to fine-tune T cell activation from aAPC by patterning activating ligands in ways that mimic T cell-APC interaction.…”