The recognition events that mediate adaptive cellular immunity and regulate antibody responses depend on intercellular contacts between T cells and antigen presenting cells (APC)1. T cell signaling is initiated at these contacts when surface-expressed antigen receptors (TCR) recognize peptide fragments (antigens) of pathogens bound to Major Histocompatibility Complex molecules (pMHC) on APCs. This, along with engagement of adhesion receptors, leads to the formation of a specialized junction between T cells and APCs, known as the immunological synapse (IS)3, which mediates efficient delivery of effector molecules and intercellular signals across the synaptic cleft2. T cell recognition of pMHC and the adhesion ligand Intercellular Adhesion Molecule-1 (ICAM-1) on supported planar bilayers recapitulates the domain organization of the immunological synapse (IS)4–5, which is characterized by central accumulation of TCR5, adjacent to a secretory domain3, both surrounded by an adhesive ring4–5. Although accumulation of TCR at the IS center correlates with T cell function4, this domain is itself largely devoid of TCR signaling activity5–6, and is characterized by an unexplained immobilization of TCR-pMHC complexes relative to the highly dynamic IS periphery4–5. Here we show that centrally accumulated TCR is located on the surface of extracellular microvesicles that bud at the IS center. Tumor susceptibility gene 101 (TSG101)6 sorts TCR for inclusion in microvesicles, while vacuolar protein sorting 4 (VPS4) 7–8 mediates scission of microvesicles from the T cell plasma membrane. The HIV polyprotein GAG co-opts this process for budding of virus-like particles. B cells bearing cognate pMHC receive TCR from T cells and initiate intracellular signals in response to isolated synaptic microvesicles. We conclude that the immunological synapse orchestrates TCR sorting and release in extracellular microvesicles. These microvesicles deliver transcellular signals across antigen-dependent synapses by engaging cognate pMHC on APC.

SUMMARY The immunological synapse formed between a cytotoxic T lymphocyte (CTL) and an infected or transformed target cell is a physically active structure capable of exerting mechanical force. Here, we investigated whether synaptic forces promote the destruction of target cells. CTLs kill by secreting toxic proteases and the pore forming protein perforin into the synapse. Biophysical experiments revealed a striking correlation between the magnitude of force exertion across the synapse and the speed of perforin pore formation on the target cell, implying that force potentiates cytotoxicity by enhancing perforin activity. Consistent with this interpretation, we found that increasing target cell tension augmented pore formation by perforin and killing by CTLs. Our data also indicate that CTLs coordinate perforin release and force exertion in space and time. These results reveal an unappreciated physical dimension to lymphocyte function and demonstrate that cells use mechanical forces to control the activity of outgoing chemical signals.

Mechanical forces play an increasingly recognized role in modulating cell function. This report demonstrates mechanosensing by T cells, using polyacrylamide gels presenting ligands to CD3 and CD28. Naive CD4 T cells exhibited stronger activation, as measured by attachment and secretion of IL-2, with increasing substrate elastic modulus over the range of 10-200 kPa. By presenting these ligands on different surfaces, this report further demonstrates that mechanosensing is more strongly associated with CD3 rather than CD28 signaling. Finally, phospho-specific staining for Zap70 and Src family kinase proteins suggests that sensing of substrate rigidity occurs at least in part by processes downstream of T-cell receptor activation. The ability of T cells to quantitatively respond to substrate rigidly provides an intriguing new model for mechanobiology.

Adoptive immunotherapy using cultured T cells holds promise for the treatment of cancer and infectious disease. Ligands immobilized on surfaces fabricated from hard materials such as polystyrene plastic are commonly employed for T cell culture. The mechanical properties of a culture surface can influence the adhesion, proliferation, and differentiation of stem cells and fibroblasts. We therefore explored the impact of culture substrate stiffness on the ex vivo activation and expansion of human T cells. We describe a simple system for the stimulation of the TCR/CD3 complex and the CD28 receptor using substrates with variable rigidity manufactured from poly(dimethylsiloxane) (PDMS), a biocompatible silicone elastomer. We show that softer (Young’s Modulus [E] < 100 kPa) substrates stimulate an average 4-fold greater IL-2 production and ex vivo proliferation of human CD4+ and CD8+ T cells compared with stiffer substrates (E >2 MPa). Mixed peripheral blood T cells cultured on the stiffer substrates also demonstrate a trend (non-significant) towards a greater proportion of CD62Lneg, effector-differentiated CD4+ and CD8+ T cells. Naïve CD4+ T cells expanded on softer substrates yield an average 3-fold greater proportion of IFN-γ producing TH1-like cells. These results reveal that the rigidity of the substrate used to immobilize T cell stimulatory ligands is an important and previously unrecognized parameter influencing T cell activation, proliferation and TH differentiation. Substrate rigidity should therefore be a consideration in the development of T cell culture systems as well as when interpreting results of T cell activation based upon solid-phase immobilization of TCR/CD3 and CD28 ligands.

Mechanical forces have key roles in regulating activation of T cells and coordination of the adaptive immune response. A recent example is the ability of T cells to sense the rigidity of an underlying substrate through the T-cell receptor (TCR) coreceptor CD3 and CD28, a costimulation signal essential for cell activation. In this report, we show that these two receptor systems provide complementary functions in regulating the cellular forces needed to test the mechanical properties of the extracellular environment. Traction force microscopy was carried out on primary human cells interacting with micrometer-scale elastomer pillar arrays presenting activation antibodies to CD3 and/or CD28. T cells generated traction forces of 100 pN on arrays with both antibodies. By providing one antibody or the other in solution instead of on the pillars, we show that force generation is associated with CD3 and the TCR complex. Engagement of CD28 increases traction forces associated with CD3 through the signaling pathway involving PI3K, rather than providing additional coupling between the cell and surface. Force generation is concentrated to the cell periphery and associated with molecular complexes containing phosphorylated Pyk2, suggesting that T cells use processes that share features with integrin signaling in force generation. Finally, the ability of T cells to apply forces through the TCR itself, rather than the CD3 coreceptor, was tested. Mouse cells expressing the 5C.C7 TCR exerted traction forces on pillars presenting peptide-loaded MHCs that were similar to those with α-CD3, suggesting that forces are applied to antigen-presenting cells during activation.T -cell activation is a key regulatory point of the adaptive immune response. It is initiated by recognition of peptideloaded MHCs (pMHCs) on antigen-presenting cells (APCs) by T-cell receptors (TCRs). Engagement of additional receptors on the T-cell surface leads to formation of a specialized interface termed the immune synapse (IS), which focuses communication between these cells. The IS has emerged as a compelling model of juxtacrine signaling, providing key insights into how the dynamics of such interfaces influence cell-cell communication. Mechanical forces originating from a range of sources, including cytoskeletal dynamics, also play important roles in T-cell activation. The initial spreading of T cells following contact with an activating surface is dependent on a burst of actin polymerization (1, 2). Subsequent retrograde flow of actin and contraction of actomyosin structures drive microscale reorganization of signaling complexes within this interface, resulting in formation of concentric central, peripheral, and distal supramolecular activation cluster (cSMAC, pSMAC, and dSMAC) structures that comprise the archetypal IS (3-8). The TCR complex itself may be triggered by external forces (9, 10), whereas TCR ligation may induce actin polymerization and generation of protrusive forces (11).More recently, mechanosensing by T cells was demonstrated in the context ...