The study objective was to establish The American Association for Thoracic Surgery (AATS) evidence-based guidelines for the management of empyema.

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 ...

Background-Neoadjuvant chemoradiotherapy followed by esophagogastrectomy has become the standard of care for patients with locally advanced esophageal cancer. This report analyzes our experience with this treatment approach.

Spatial organization of signaling complexes is a defining characteristic of the immunological synapse (IS), but its impact on cell communication is unclear. In T cell-APC pairs, more IL-2 is produced when CD28 clusters are segregated from central supramolecular activation cluster (cSMAC)-localized CD3 and into the IS periphery. However, it is not clear in these cellular experiments whether the increased IL-2 is driven by the pattern itself or by upstream events that precipitate the patterns. In this article, we recapitulate key features of physiological synapses using planar costimulation arrays containing antibodies against CD3 and CD28, surrounded by ICAM-1, created by combining multiple rounds of microcontact printing on a single surface. Naïve T cells traverse these arrays, stopping at features of anti-CD3 antibodies and forming a stable synapse. We directly demonstrate that presenting anti-CD28 in the cell periphery, surrounding an anti-CD3 feature, enhances IL-2 secretion by naïve CD4 ؉ T cells compared with having these signals combined in the center of the IS. This increased cytokine production correlates with NF-B translocation and requires PKB/Akt signaling. The ability to arbitrarily and independently control the locations of anti-CD3 and anti-CD28 offered the opportunity to examine patterns not precisely attainable in cell-cell interfaces. With these patterns, we show that the peripheral presentation of CD28 has a larger impact on IL-2 secretion than CD3 colocalization/ segregation.costimulation ͉ immunology ͉ naïve T cells ͉ microarrays C ommunication between T cells and antigen-presenting cells (APCs) forms a key regulatory point of the immune system, mediated in large part by interactions between cell-surface proteins in a small (Ϸ75 m 2 ) intercellular contact area termed the immunological synapse (IS). It is hypothesized that the spatial organization of receptor/ligand complexes within the IS is an important part of the language of T cell-APC communication (1, 2). The archetypical organization of mature synapses consists of a central supramolecular activation cluster (cSMAC) containing TCR/MHC complexes and surrounded by a peripheral (pSMAC) region rich in ICAM-1/LFA-1 clusters; the function of this bull's-eye pattern, as well as alternative geometries, in modulating T cell activation is of much contemporary interest (3-7). Recent studies have also suggested a functional role of microscale organization of CD28 ligation, a classic costimulatory signal, in modulating T cell response. Andres et al. (8) showed that during the initial minutes of T cell/APC interaction, CD28 and TCR comigrate, leading to cSMAC localization of both complexes. At later time points, Tseng and colleagues (9) showed that CD28/CD80 complexes segregate from TCR/MHC into either the cSMAC periphery or pSMAC. Surprisingly, truncation of the CD80 cytosolic domain, which reduces T cell activation (10), promoted colocalization of TCR/MHC and CD28/CD80 complexes in the cSMAC (9). Because the modifications to CD80 were in the APC, ...