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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.
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.
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.
Cytotoxic T lymphocytes (CTLs) are involved in development of diabetes. However, the impact of excessive glucose on CTL-mediated antigen-independent killing remains elusive. Here, we report that TNF-related apoptosis inducing ligand (TRAIL) is substantially up- regulated in CTLs in environments with high glucose (HG) both in vitro and in vivo. The PI3K- Akt-NFκB axis and non-mitochondrial reactive oxygen species are essential in HG-induced TRAIL upregulation in CTLs. TRAILhigh CTLs induce apoptosis of pancreatic beta cell line 1.4E7. Metformin and Vitamin D synergistically reduce HG-enhanced expression of TRAIL in CTLs and coherently protect 1.4E7 cells from TRAIL-mediated apoptosis. Notably, in patients with diabetes, correlation between Vitamin D concentrations in plasma and glucose levels is linked to HG-enhanced TRAIL expression on CTLs. Microarray data reveal that OXCT2, an important enzyme in ketone body catabolism, is a promising target in response to vitamin D. Our work not only reveals a novel mechanism of CTL involvement in progression of diabetes, but also establishes CTLs as a target for combined metformin and vitamin D therapy to protect pancreatic beta cells of diabetic patients.Competing Interest StatementThe authors have declared no competing interest.
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.
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.
