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- ItemCharacterization of immune cell migration using microfabrication(Berlin ; Heidelberg : Springer, 2021) Vesperini, Doriane; Montalvo, Galia; Qu, Bin; Lautenschläger, FranziskaThe immune system provides our defense against pathogens and aberrant cells, including tumorigenic and infected cells. Motility is one of the fundamental characteristics that enable immune cells to find invading pathogens, control tissue damage, and eliminate primary developing tumors, even in the absence of external treatments. These processes are termed “immune surveillance.” Migration disorders of immune cells are related to autoimmune diseases, chronic inflammation, and tumor evasion. It is therefore essential to characterize immune cell motility in different physiologically and pathologically relevant scenarios to understand the regulatory mechanisms of functionality of immune responses. This review is focused on immune cell migration, to define the underlying mechanisms and the corresponding investigative approaches. We highlight the challenges that immune cells encounter in vivo, and the microfabrication methods to mimic particular aspects of their microenvironment. We discuss the advantages and disadvantages of the proposed tools, and provide information on how to access them. Furthermore, we summarize the directional cues that regulate individual immune cell migration, and discuss the behavior of immune cells in a complex environment composed of multiple directional cues.
- ItemCharacterization of the Elasticity of CD4+ T Cells: An Approach Based on Peak Force Quantitative Nanomechanical Mapping(Sunnyvale, CA : bio-protocol.org, 2022) Jung, Philipp; Zhou, Xiangda; Iden, Sandra; Qu, Bin; Bischoff, MarkusCD4+ T cells are essential players in orchestrating the specific immune response against intracellular pathogens, and in inhibiting tumor development in an early stage. The activation of T cells is triggered by engagement of T cell receptors (TCRs). Here, CD3 and CD28 molecules are key factors, (co)stimulating signaling pathways essential for activation and proliferation of CD4+ T cells. T cell activation induces the formation of a tight mechanical bond between T cell and target cell, the so-called immunological synapse (IS). Due to this, mechanical cell properties, including stiffness, play a significant role in modulating cell functions. In the past, many approaches were made to investigate mechanical properties of immune cells, including micropipette aspiration, microplate-based rheometry, techniques based on deformation during cytometry, or the use of optical tweezers. However, the stiffness of T lymphocytes at a subcellular level at the IS still remains largely elusive. With this protocol, we introduce a method based on atomic force microscopy (AFM), to investigate the local cellular stiffness of T cells on functionalized glass/Polydimethylsiloxan (PDMS) surfaces, which mimicks focal stimulation of target cells inducing IS formation by T cells. By applying the peak force nanomechanical mapping (QNM) technique, cellular surface structures and the local stiffness are determined simultaneously, with a resolution of approximately 60 nm. This protocol can be easily adapted to investigate the mechanical impact of numerous factors influencing IS formation and T cell activation.
- ItemHigh glucose distinctively regulates Ca2+ influx in cytotoxic T lymphocytes upon target recognition and thapsigargin stimulation(Hoboken, NJ : Wiley, 2020) Zou, Huajiao; Yang, Wenjuan; Schwär, Gertrud; Zhao, Renping; Alansary, Dalia; Yin, Deling; Schwarz, Eva C.; Niemeyer, Barbara A.; Qu, BinIn CTLs: High glucose‐culture enhances thapsigargin‐induced SOCE but decreases target recognition‐induced Ca2+ influx. High glucose‐culture regulates expression of ORAIs and STIMs without affecting glucose uptake. More high glucose‐cultured CTLs are prone to necrosis after execution of killing.