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Efficacy of cytotoxic T lymphocyte (CTL)-based immunotherapy is still unsatisfactory against solid tumors, which are frequently characterized by condensed extracellular matrix. Here, using a unique 3D killing assay, we identify that the killing efficiency of primary human CTLs is substantially impaired in dense collagen matrices. Although the expression of cytotoxic proteins in CTLs remained intact in dense collagen, CTL motility was largely compromised. Using light-sheet microscopy, we found that persistence and velocity of CTL migration was influenced by the stiffness and porosity of the 3D matrix. Notably, 3D CTL velocity was strongly correlated with their nuclear deformability, which was enhanced by disruption of the microtubule network especially in dense matrices. Concomitantly, CTL migration, search efficiency, and killing efficiency in dense collagen were significantly increased in microtubule-perturbed CTLs. In addition, the chemotherapeutically used microtubule inhibitor vinblastine drastically enhanced CTL killing efficiency in dense collagen. Together, our findings suggest targeting the microtubule network as a promising strategy to enhance efficacy of CTL-based immunotherapy against solid tumors, especially stiff solid tumors.
Cytotoxic T lymphocytes (CTLs) are key players to eliminate tumorigenic or pathogen-infected cells using lytic granules (LG) and Fas ligand (FasL) pathways. Depletion of glucose leads to severely impaired cytotoxic function of CTLs. However, the impact of excessive glucose on CTL functions still remains largely unknown. Here we used primary human CD8(+) T cells, which were stimulated by CD3/CD28 beads and cultured in medium either containing high glucose (HG, 25 mM) or normal glucose (NG, 5.6 mM). We found that in HG-CTLs, glucose uptake and glycolysis were enhanced, whereas proliferation remained unaltered. Furthermore, CTLs cultured in HG exhibited an enhanced CTL killing efficiency compared to their counterparts in NG. Unexpectedly, expression of cytotoxic proteins (perforin, granzyme A, granzyme B and FasL), LG release, cytokine/cytotoxic protein release and CTL migration remained unchanged in HG-cultured CTLs. Interestingly, additional extracellular Ca2+ diminished HG-enhanced CTL killing function. Our findings suggest that in an environment with excessive glucose, CTLs could eliminate target cells more efficiently, at least for a certain period of time, in a Ca2+-dependent manner.
Progress in our understanding of mechanotransduction events requires noninvasive methods for the manipulation of forces at molecular scale in physiological environments. Inspired by cellular mechanisms for force application (i.e. motor proteins pulling on cytoskeletal fibers), we present a unique molecular machine that can apply forces at cell-matrix and cell-cell junctions using light as an energy source. The key actuator is a light-driven rotatory molecular motor linked to polymer chains, which is intercalated between a membrane receptor and an engineered biointerface. The light-driven actuation of the molecular motor is converted in mechanical twisting of the entangled polymer chains, which will in turn effectively “pull” on engaged cell membrane receptors (e.g., integrins, T cell receptors) within the illuminated area. Applied forces have physiologically-relevant magnitude and occur at time scales within the relevant ranges for mechanotransduction at cell-friendly exposure conditions, as demonstrated in force-dependent focal adhesion maturation and T cell activation experiments. Our results reveal the potential of nanomotors for the manipulation of living cells at the molecular scale and demonstrate a functionality which at the moment cannot be achieved by other technologies for force application.
Collagen density defines 3D migration of CTLs and their consequent cytotoxicity against tumor cells
(2021)
Solid tumors are often characterized by condensed extracellular matrix (ECM). The impact of dense ECM on cytotoxic T lymphocytes (CTL) function is not fully understood. Here, we report that CTL-mediated cytotoxicity is substantially impaired in dense collagen matrices. Although the intrinsic killing machinery including expression of cytotoxic proteins and degranulation was intact, CTL motility was substantially compromised in dense collagen. We found that for 3D CTL migration, persistence and velocity was regulated by collagen stiffness and the porosity, respectively. Interestingly, 3D CTL velocity is strongly correlated with their nuclear deformability/flexibility during migration, which is regulated by the microtubule network. Moreover, CTL migration was completely abolished by inhibition of actin polymerization and or myosin IIA. Remarkably, disruption of the microtubule-networks significantly improves the impaired migration, search efficiency, and cytotoxicity of CTLs in dense collagen. Our work suggests the microtubule network as a promising target to rescue impaired CTL killing capacity in solid tumor related scenarios.
Profiling of cytokines, chemokines and growth factors in saliva and gingival crevicular fluid
(2021)
In saliva and gingival crevicular fluid (GCF) soluble factors such as cytokines, chemokines and growth factors have shown a great potential serving as biomarkers for early detection and/or diagnosis of oral and systemic diseases. However, GCF and saliva, which one is a better source is still under debate. This study aimed to gain an overview of cytokines, chemokines and growth factors in saliva and GCF to pave the way for selecting suitable oral fluids for oral and systemic diseases. Multiplex cytokine assay was conducted to determine concentrations of cytokines, chemokines and growth factors in saliva and GCF samples from healthy subjects. The protocol for sample collection was carefully optimized. Stabilization, repeatability, and donor variation of the profiles were analyzed. We found that for different donors, cytokine and chemokine profiles showed unique patterns in saliva but similar patterns in GCF. In terms of growth factors, the profiles were individualized in saliva and GCF. All profiles stayed stable for the same healthy individual. In saliva, profiles of cytokines, chemokines and growth factors are individualized for different donors. In GCF, profiles of cytokines and chemokines are similar. Other factors, such as growth factors and T helper-related cytokines, are highly variable in donors. Profiles of soluble factors are not correlated in saliva and GCF. The comprehensive cytokine profiles in saliva and GCF reported in this work would serve as a good base for choosing promising cytokines for developing biomarkers in oral fluids.Competing Interest StatementThe authors have declared no competing interest.
Effective T cell responses against tumor cells require diverse effector functions including polarization towards tumor cells to form immunological synapses and nuclear factor of activated T-cells (NFAT)-dependent gene transcription. While the role of tumor cell softening has been associated with malignancy, stemness, and metastasis, potentially contributing to immune evasion, its impact on cellular processes in T cells is not well understood. Here, we show that both T cell polarization and NFAT nuclear translocation are modulated by target stiffness in a Ca2+ dependent manner. Using both anti-CD3 antibody-functionalized substrates with varying stiffness as surrogates for target cells or softened tumor cells, we found that both, reorientation of microtubule organizing center (MTOC) towards the tumor cells, a hallmark for T cell polarization, and NFAT translocation were impaired on softer hydrogels or following contact with softer cancer cells. The amplitudes of intracellular Ca2+ signals were dependent on stiffness, and removal of extracellular Ca2+ inhibited stiffness-dependent T cell responsiveness. While stiffness-dependent Ca2+ signaling was crucial for both, T cell polarization and NFAT translocation, Ca2+ influx through Piezo1, a mechanosensitive ion channel, mediated stiffness-dependent MTOC reorientation but not NFAT translocation. In contrast, Ca2+ influx through store-operated Orai channels mediated NFAT translocation but not MTOC reorientation. Our results demonstrate that tumor cell stiffness directly influences T cell functionality through distinct Ca2+ influx pathways, revealing cell softening as an essential mechanism employed by malignant cells to evade immune surveillance.
TNF-related apoptosis inducing ligand (TRAIL) is expressed on cytotoxic T lymphocytes (CTLs) and TRAIL is linked to progression of diabetes. However, the impact of high glucose on TRAIL expression and its related killing function in CTLs still remains largely elusive. Here, we report that TRAIL is substantially up-regulated in CTLs in environments with high glucose (HG) both in vitro and in vivo. Non-mitochondrial reactive oxygen species, NFκB and PI3K/Akt are essential in HG-induced TRAIL upregulation in CTLs. TRAIL<sup>high</sup> CTLs induce apoptosis of pancreatic beta cell line 1.4E7. Treatment with metformin and vitamin D reduces HG-enhanced expression of TRAIL in CTLs and coherently protects 1.4E7 cells from TRAIL-mediated apoptosis. Our work suggests that HG-induced TRAIL<sup>high</sup> CTLs might contribute to the destruction of pancreatic beta cells in a hyperglycemia condition.
Visualizing interactions between cells and the extracellular matrix (ECM) mesh is important to understand cell behavior and regulatory mechanisms by the extracellular environment. However, long term visualization of three-dimensional (3D) matrix structures remains challenging mainly due to photobleaching or blind spots perpendicular to the imaging plane. Here, we combine label-free light-sheet scattering microcopy (LSSM) and fluorescence microscopy to solve these problems. We verified that LSSM can reliably visualize structures of collagen matrices from different origin including bovine, human and rat tail. The quality and intensity of collagen structure images acquired by LSSM did not decline with time. LSSM offers abundant wavelength choice to visualize matrix structures, maximizing combination possibilities with fluorescently-labelled cells, allowing visualizing of long-term ECM-cell interactions in 3D. Interestingly, we observed ultrathin thread-like structures between cells and matrix using LSSM, which were not observed by normal fluorescence microscopy. Transient local alignment of matrix by cell-applied forces can be observed. In summary, LSSM provides a powerful and robust approach to investigate the complex interplay between cells and ECM.
Background Natural killer (NK) cells play a key role in eliminating tumorigenic and pathogen-infected cells. Verbena officinalis (V. officinalis) has been used as a medical plant in traditional and modern medicine, exhibiting anti-tumor and anti-inflammation activity.Purpose The impact of bioactive constituents of V. officinalis on immune responses still remains largely elusive. In this work we investigated the potential targets of V. officinalis and focused on killing efficiency and related functions of NK cells regulated by bioactive constituents of V. officinalis.Study design/methods We used primary human NK cells from peripheral blood mononuclear cells. Potential regulatory roles of selected compounds were analyzed by network pharmacology approaches. Killing efficiency was determined with real-time killing assay and live-cell imaging in 3D. Proliferation was examined by CFSE staining. Expression of cytotoxic proteins was analyzed using flow cytometry. Lytic granule release was quantified by CD107a degranulation assay. Contact time required for killing and determination of serial killers were analyzed using live cell imaging results. Results: Using network pharmacology approaches, we analyzed potential regulatory roles of five compounds (Acteoside, Apigenin, Kaempferol, Verbenalin and Hastatoside) from V. officinalis on immune cell functions and revealed NK cells as a major target. The effect of these compounds on NK killing efficiency was examined with real-time killing assay, and Verbenalin enhanced NK killing efficiency significantly. Further investigation showed that Verbenalin did not affect proliferation, expression of cytotoxic proteins, or lytic granule degranulation, but rather reduced contact time required for killing therefore enhancing total killing events per NK cell, suggestively via inhibition of inhibitory receptors as determined by docking assay.Conclusions Our findings reveal the underlying mechanisms how V. officinalis regulates functions of immune cells, especially NK cells, suggesting Verbenalin from V. officinalis as a promising therapeutic reagent to fight cancer and infection.Competing Interest StatementThe authors have declared no competing interest.
