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 ...

Polarization of the T cell microtubule-organizing center (MTOC) toward the antigen-presenting cell is driven by the accumulation of diacylglycerol at the immunological synapse (IS). The mechanisms that couple diacylglycerol to the MTOC are not known. Using single-cell photoactivation of the T cell receptor, we demonstrated that three distinct protein kinase C (PKC) isoforms are recruited by diacylglycerol to the IS in two steps. PKC-ε and PKC-η accumulated first in a broad region of membrane, while PKC-θ arrived later in a smaller zone. Functional experiments indicated that PKC-θ was required for MTOC reorientation, and that PKC-ε and PKC-η operated redundantly to promote PKC-θ recruitment and subsequent polarization responses. These results establish a previously uncharacterized role for PKCs in T cell polarity.

Phylogenetic analyses have provided strong evidence that amino acid changes in spike (S) protein of animal and human SARS coronaviruses (SARS-CoVs) during and between two zoonotic transfers (2002/03 and 2003/04) are the result of positive selection. While several studies support that some amino acid changes between animal and human viruses are the result of inter-species adaptation, the role of neutralizing antibodies (nAbs) in driving SARS-CoV evolution, particularly during intra-species transmission, is unknown. A detailed examination of SARS-CoV infected animal and human convalescent sera could provide evidence of nAb pressure which, if found, may lead to strategies to effectively block virus evolution pathways by broadening the activity of nAbs. Here we show, by focusing on a dominant neutralization epitope, that contemporaneous- and cross-strain nAb responses against SARS-CoV spike protein exist during natural infection. In vitro immune pressure on this epitope using 2002/03 strain-specific nAb 80R recapitulated a dominant escape mutation that was present in all 2003/04 animal and human viruses. Strategies to block this nAb escape/naturally occurring evolution pathway by generating broad nAbs (BnAbs) with activity against 80R escape mutants and both 2002/03 and 2003/04 strains were explored. Structure-based amino acid changes in an activation-induced cytidine deaminase (AID) “hot spot” in a light chain CDR (complementarity determining region) alone, introduced through shuffling of naturally occurring non-immune human VL chain repertoire or by targeted mutagenesis, were successful in generating these BnAbs. These results demonstrate that nAb-mediated immune pressure is likely a driving force for positive selection during intra-species transmission of SARS-CoV. Somatic hypermutation (SHM) of a single VL CDR can markedly broaden the activity of a strain-specific nAb. The strategies investigated in this study, in particular the use of structural information in combination of chain-shuffling as well as hot-spot CDR mutagenesis, can be exploited to broaden neutralization activity, to improve anti-viral nAb therapies, and directly manipulate virus evolution.

Centrosome reorientation to the immunological synapse maintains the specificity of T-cell effector function by facilitating the directional release of cytokines and cytolytic factors toward the antigen-presenting cell. This polarization response is driven by the localized accumulation of diacylglycerol, which recruits multiple protein kinase (PK)C isozymes to the synaptic membrane. Here, we used T-cell receptor (TCR) photoactivation and imaging methodology to demonstrate that PKCs control centrosome dynamics through the reciprocal localization of two motor complexes, dynein and nonmuscle myosin (NM)II. Dynein accumulated in the region of TCR stimulation, whereas NMII clustered in the back of the cell, behind the polarizing centrosome. PKC activity, which shaped both dynein and NMII accumulation within this framework, controlled NMII localization directly by phosphorylating inhibitory sites within the myosin regulatory light chain, thereby suppressing NMII clustering in the region of TCR stimulation. Concurrently, phosphorylation of distinct sites within myosin regulatory light chain by Rho kinase drove NMII clustering in areas behind the centrosome. These results reveal a role for NMII in T-cell polarity and demonstrate how it is regulated by upstream signals.cytoskeleton | polarity | signal transduction | microtubule-organizing center