Patterned protein surfaces that modulate immunological synapse structure formation in T cells

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2006.Vita.Includes bibliographical references (leaves 128-137).T cells are activated by recognition of foreign peptides displayed on the surface of antigen presenting cells (APCs), an event that triggers assembly of a complex microscale structure at the T cell-APC interface known as the immunological synapse (IS). It remains unresolved whether the unique physical structure of the synapse itself impacts the functional response of T cells, independent of the quantity and quality of ligands encountered by the T cell. As a first step toward addressing this question, we fabricated multicomponent protein surfaces that surrogate the role of APCs and studied T cell responses as a function of synapse structure. To pattern multiple proteins on surfaces, we synthesized and characterized a new polymer, poly(o-ntrobenzyl methacrylate-r-methyl methacrylate-poly(ethylene glycol) methacrylate (PNMP), a photoresist that can be processed under mild aqueous conditions. Based on the pH- and temperature-sensitive solubility of UV-exposed PNMP random terpolymers in aqueous buffers, two-component protein patterning was achieved under conditions that avoid exposing proteins to conditions outside the narrow range of physiological pH, ionic strength, and temperature where their stability is greatest.(cont.) Using a photolithographic strategy we developed employing this novel PNMP photoresist polymer, we created multicomponent protein surfaces presenting micron-scale arrays of tethered T cell receptor (TCR) ligands (anti-CD3 'activation sites') surrounded by a field of tethered intercellular adhesion molecule-I (ICAM-1), as a model substrate on which T cells could be seeded to mimic T cell-APC interactions. CD4+ T cells seeded on these surfaces polarized and migrated; on contact with activation sites, T cells assembled an IS with a structure modulated by the physical pattern of ligand encountered. On surfaces patterned with focal spots of TCR ligand, T cells stably interacted with activation sites, proliferated, and secreted cytokines. In contrast, T cells interacting with activation sites patterned to preclude centralized clustering of TCR ligand failed to form stable contacts with activation sites, exhibited aberrant PKC-[Theta] clustering in a fraction of cells, and had significantly reduced production of interferon-[gamma]. These results suggest that focal clustering of TCR ligand characteristic of the 'mature' IS may be required under some conditions for full T cell activation.by Junsang Doh.Ph.D

T cell migration in microchannels densely packed with T cells

T cells migrate diverse microenvironments of the body to mount antigen-specific immune responses. T cell activation, a key initial process for antigen-specific immune responses, occur in secondary lymphoid organs such as spleens and lymph nodes where high density of T cells migrates rapidly through the reticular networks formed by stromal cells. In vitro model system recapitulating key characteristics of secondary lymphoid organs, confined spaces densely packed with rapidly migrating cells, would be useful to investigate mechanisms ofT cell migration. In this study, we devised a method to fabricate microchannels densely packed with T cells. Microchannel arrays with fixed height (4 mu m) and length (1.5 mm) and various widths (15 similar to 80 mu m) were fabricated in between trapezoid-shaped reservoirs that facilitated T cell sedimentation near microchannel entries. Microchannel surface chemistry and filling time were optimized to achieve high packing density (0.89) of T cell filling within microchannels. Particle image velocimetry (PIV) analysis method was employed to extract velocity field of microchannels densely packed with T cells. Using velocity field information, various motility parameters were further evaluated to quantitatively assess the effects of microchannel width and media tonicity on T cell motility within cell dense microenvironments.Y

Hybrid bright-field and hologram imaging of cell dynamics

Volumetric observation is essential for understanding the details of complex biological phenomena. In this study, a bright-field microscope, which provides information on a specific 2D plane, and a holographic microscope, which provides information spread over 3D volumes, are integrated to acquire two complementary images simultaneously. The developed system was successfully applied to capture distinct T-cell adhesion dynamics on inflamed endothelial layers, including capture, rolling, crawling, transendothelial migration, and subendothelial migration.113Ysciescopu

Turning behaviors of T cells climbing up ramp-like structures are regulated by myosin light chain kinase activity and lamellipodia formation

T cells navigate diverse microenvironments to perform immune responses. Micro-scale topographical structures within the tissues, which may inherently exist in normal tissues or may be formed by inflammation or injury, can influence T cell migration, but how T cell migration is affected by such topographical structures have not been investigated. In this study, we fabricated ramp-like structures with a 5 mu m height and various slopes, and observed T cells climbing up the ramp-like structures. T cells encountering the ramp-like structures exhibited MLC accumulation near head-tail junctions contacting the ramp-like structures, and made turns to the direction perpendicular to the ramp-like structures. Pharmacological study revealed that lamellipodia formation mediated by arp2/3 and contractility regulated by myosin light chain kinase (MLCK) were responsible for the intriguing turning behavior of T cells climbing the ramp-like structures. Arp2/3 or MLCK inhibition substantially reduced probability of T cells climbing sharp-edged ramp-like structures, indicating intriguing turning behavior of T cells mediated by lamellipodia formation and MLCK activity may be important for T cells to access inflamed or injured tissues with abrupt topographical changes.11Ysciescopu

Microfluidic system for monitoring temporal variations of hemorheological properties and platelet adhesion in LPS-injected rats

Sepsis causes multiple organs failures and eventually death. Changes in blood constituents due to sepsis lead to alterations in hemorheological properties, and cell adhesiveness. In this study, a new microfluidic system is proposed to measure temporal variations in biophysical properties of blood after injecting lipopolysaccharide (LPS) into a rat extracorporeal model under ex vivo condition. To measure blood viscosity, the interfacial line between blood and a reference fluid is formed in a Y-shaped channel. Based on the relation between interfacial width and pressure ratio, the temporal variation in blood viscosity is estimated. Optical images of blood flows are analyzed by decreasing flow rate for examination of red blood cell (RBC) aggregation. Platelets initiated by shear acceleration around the stenosis adhere to the post-stenosed region. By applying a correlation map that visualizes the decorrelation of the streaming blood flow, the area of adhered platelets can be quantitatively attained without labeling of platelets. To assess sepsis inflammation, conventional biomarkers (PCT and IL-8) are also monitored. The increasing tendency for blood viscosity, RBC aggregation, platelet adhesion, and septic biomarkers are observed after LPS injection. This microfluidic system would be beneficial for monitoring the changes in hemorheological properties and platelet activation caused by sepsis.116Ysciescopu

Anti-tumor effects of NK cells and anti-PD-L1 antibody with antibody-dependent cellular cytotoxicity in PD-L1-positive cancer cell lines

Background Although programmed cell death-1/programmed death-ligand 1 (PD-L1) inhibitors show remarkable antitumor activity, a large portion of patients with cancer, even those with high PD-L1-expressing tumors, do not respond to their effects. Most PD-L1 inhibitors contain modified fragment crystallizable region (Fc) receptor binding sites to prevent antibody-dependent cellular cytotoxicity (ADCC) against PD-L1-expressing non-tumor cells. However, natural killer (NK) cells have specific antitumor activity in the presence of tumor-targeting antibody through ADCC, which could enhance NK cell-induced cytotoxicity. We evaluated the antitumor efficacy of ADCC via anti-PD-L1 monoclonal antibodies (mAbs) and NK cells against several PD-L1-positive cancer cell lines. Methods Various cancer cell lines were used as target cell lines. Surface PD-L1 expression was analyzed by flow cytometry. IMC-001 and anti-hPD-L1-hIgG1 were tested as anti-PD-L1 mAbs with ADCC and atezolizumab as an anti-PD-L1 mAb without ADCC. NK cell cytotoxicity was measured by(51)Cr-release assay and CD107a degranulation assay. Also, live cell imaging was performed to evaluate cytotoxicity in a single-cell level. NK-92-CD16 (CD16-transduced NK-92 cell line) and peripheral blood mononuclear cells from healthy donors, respectively, were used as an effector cell. Fc gamma RIIIa (CD16a)-V158F genotyping was performed for healthy donors. Results We demonstrated that the cytotoxicity of NK-92-CD16 cells toward PD-L1-positive cancer cell lines was significantly enhanced in the presence of anti-PD-L1 mAb with ADCC. We also noted a significant increase in primary human NK cell cytotoxicity against PD-L1-positive human cancer cells when cocultured with anti-PD-L1 mAb with ADCC. Moreover, NK cells expressing aFCGR3Ahigh-affinity genotype displayed higher anti-PD-L1 mAb-mediated ADCC lysis of tumor cells than donors with a low-affinity genotype. Conclusion These results suggest that NK cells induce an ADCC response in combination with anti-PD-L1 mAbs, which helps promote ADCC antitumor activity against PD-L1-positive tumors. This study provides support for NK cell immunotherapy against high PD-L1-expressing tumors in combination with ADCC through anti-PD-L1 mAbs.

MicroRNA-150 modulates intracellular Ca2+ levels in naïve CD8+ T cells by targeting TMEM20

Regulation of intracellular Ca2+ signaling is a major determinant of CD8+ T cell responsiveness, but the mechanisms underlying this regulation of Ca2+ levels, especially in naïve CD8+ T cells, are not fully defined. Here, we showed that microRNA-150 (miR-150) controls intracellular Ca2+ levels in naïve CD8+ T cells required for activation by suppressing TMEM20, a negative regulator of Ca2+ extrusion. miR-150 deficiency increased TMEM20 expression, which resulted in increased intracellular Ca2+ levels in naïve CD8+ T cells. The subsequent increase in Ca2+ levels induced expression of anergy-inducing genes, such as Cbl-b, Egr2, and p27, through activation of NFAT1, as well as reduced cell proliferation, cytokine production, and the antitumor activity of CD8+ T cells upon antigenic stimulation. The anergy-promoting molecular milieu and function induced by miR-150 deficiency were rescued by reinstatement of miR-150. Additionally, knockdown of TMEM20 in miR-150-deficient naïve CD8+ T cells reduced intracellular Ca2+ levels. Our findings revealed that miR-150 play essential roles in controlling intracellular Ca2+ level and activation in naïve CD8+ T cells, which suggest a mechanism to overcome anergy induction by the regulation of intracellular Ca2+ levels115Ysciescopu

Differentially Expressed Potassium Channels Are Associated with Function of Human Effector Memory CD8+T cells

The voltage-gated potassium channel, Kv1.3, and the Ca2+-activated potassium channel, KCa3.1, regulate membrane potentials in T cells, thereby controlling T cell activation and cytokine production. However, little is known about the expression and function of potassium channels in human effector memory ( EM) CD8+ T cells that can be further divided into functionally distinct subsets based on the expression of the interleukin ( IL)-7 receptor alpha ( IL-7R alpha) chain. Herein, we investigated the functional expression and roles of Kv1.3 and KCa3.1 in EM CD8+ T cells that express high or low levels of the IL-7 receptor alpha chain ( IL-7R alpha(high) and IL-7R alpha(low), respectively). In contrast to the significant activity of Kv1.3 and KCa3.1 in IL-7Rahigh EM CD8+ T cells, IL-7Ralow EM CD8+ T cells showed lower expression of Kv1.3 and insignificant expression of KCa3.1. Kv1.3 was involved in the modulation of cell proliferation and IL-2 production, whereas KCa3.1 affected the motility of EM CD8+ T cells. The lower motility of IL-7Ralow EM CD8+ T cells was demonstrated using transendothelial migration and motility assays with intercellular adhesion molecule 1-and/or chemokine stromal cell-derived factor-1 alpha-coated surfaces. Consistent with the lower migration property, IL-7Ralow EM CD8+ T cells were found less frequently in human skin. Stimulating IL-7Ralow EM CD8+ T cells with IL-2 or IL-15 increased their motility and recovery of KCa3.1 activity. Our findings demonstrate that Kv1.3 and KCa3.1 are differentially involved in the functions of EM CD8+ T cells. The weak expression of potassium channels in IL-7Ralow EM CD8+ T cells can be revived by stimulation with IL-2 or IL-15, which restores the associated functions. This study suggests that IL-7Rahigh EM CD8+ T cells with functional potassium channels may serve as a reservoir for effector CD8+ T cells during peripheral inflammation.112Ysciescopu