Biology subjects

Schwarz, E. C.

Publications and source records attributed to Schwarz, E. C..

6 recordsLinked to original sources

bioRxiv2021

Metabolic reprogramming of cytotoxic T lymphocytes by high glucose enhances pancreatic beta cell apoptosis via TRAIL

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-NF{kappa}B 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.

immunology↗

bioRxiv2021

Collagen density defines 3D migration of CTLs and their consequent cytotoxicity against tumor cells

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.

immunology↗

bioRxiv2021

Cytotoxic efficiency of human CD8+ T cell memory subtypes

Immunological memory is an important concept to protect humans against recurring diseases. Memory CD8+ T cells are required for quick expansion into effector cells but also provide immediate cytotoxicity against their targets. Whereas many functions of the two main cytotoxic subtypes, effector memory CD8+ T cells (TEM) and central memory CD8+ T cells (TCM), are relatively well defined, single TEM and TCM cell cytotoxicity has not been quantified. Here, we analyze human CD8+ subtype distribution following SEA stimulation and quantify the expression of death mediators, including perforin, granzyme B, FasL and TRAIL. We find higher perforin, granzyme B and FasL expression in TEM and compared to TCM. To quantify single TEM and TCM cytotoxicity, we develop a FRET-based fluorescent assay with NALM6 target cells stably transfected with a GFP-RFP FRET construct based on a caspase-cleavage sequence (apoptosis sensor Casper-GR). Applying this assay, TEM or TCM induced target cell apoptosis or necrosis can be quantified. We find that single TEM are much more effective than TCM in killing their targets mainly by apoptosis and secondary necrosis. The reason for this is the higher perforin expression and on a more efficient lytic immunological synapse during TEM-target contact compared to TCM-target contact. Defining and quantifying single TEM and TCM cytotoxicity and the respective mechanism should be helpful to optimize future subset-based immune therapies.

immunology↗

bioRxiv2020

Identification of molecular candidates which regulate calcium-dependent CD8+ T-cell cytotoxicity

Cytotoxic CD8+ T lymphocytes (CTL) eliminate infected cells or transformed tumour cells by releasing perforin-containing cytotoxic granules at the immunological synapse. The secretion of such granules depends on Ca2+-influx through store operated Ca2+ channels, formed by STIM-activated Orai proteins. Whereas molecular mechanisms of the secretion machinery are well understood, much less is known about the molecular machinery that regulates the efficiency of Ca2+-dependent target cell killing. Here, we isolated total RNA from natural killer (NK) cells, non-stimulated CD8+ T-cells, and from Staphylococcus aureus enterotoxin A (SEA) stimulated CD8+ T-cells (SEA-CTL) and conducted whole genome expression profiling by microarray experiments. Based on differential expression analysis of the transcriptome data and analysis of master regulator genes, we identified 31 candidates which potentially regulate Ca2+-homeostasis in CTL. To investigate a putative function of these candidates in CTL cytotoxicity, we transfected either SEA-stimulated CTL (SEA-CTL) or antigen specific CD8+ T-cell clones (CTL-MART-1) with siRNAs specific against the identified candidates and analyzed the killing capacity using a real-time killing assay. In addition, we complemented the analysis by studying the effect of inhibitory substances acting on the candidate proteins if available. Finally, to unmask their involvement in Ca2+ dependent cytotoxicity, candidates were also analyzed under Ca2+-limiting conditions. Overall, this strategy led to the identification of KCNN4, RCAN3, CCR5 and BCL2 as potential candidates to regulate the efficiency of Ca2+-dependent target cell killing.

immunology↗

bioRxiv2020

Combined CTL and NK cell cytotoxicity against cancer cells

CTL and NK cells recognize and eliminate cancer cells. However, immune evasion, down regulation of immune function by the tumor microenvironment, or resistance of cancer cells are a major problem. While CTL and NK cells are both important to eliminate cancer, most studies address them individually. In a new experimental human model, we analysed combined primary human CTL and NK cell cytotoxicity against the melanoma cell line SK-Mel-5. At high effector-to-target ratios, MART-1-specific CTL or NK cells eliminated SK-Mel-5 cells within 24 hours indicating that SK-Mel-5 cells are initially not resistant. However, at lower effector-to-target ratios, which resemble conditions of the immune contexture in human cancer, a significant number of SK-Mel-5 cells survived. Whereas CTL pre-exposure induced resistance in surviving SK-Mel-5 cells to subsequent CTL or NK cell cytotoxicity, NK cell pre-exposure induced resistance in surviving SK-Mel-5 cells to NK cells but not to MART-1 specific CTL. In contrast, there was even a slight enhancement of CTL cytotoxicity against SK-Mel-5 cells following NK cell pre-exposure. In all other combinations, resistance to subsequent cytotoxicity was higher, if melanoma cells were pre-exposed to larger numbers of CTL or NK cells. Increases in human leukocyte antigen class I expression correlated with resistance to NK cells, while reduction in MART-1 antigen expression correlated with reduced CTL cytotoxicity. CTL cytotoxicity was rescued beyond control levels by exogenous MART-1 antigen. This study quantifies the interdependence of CTL and NK cell cytotoxicity and may guide strategies for efficient CTL-NK cell anti-melanoma therapies. Key points summaryO_LICytotoxic T lymphocytes (CTL) and natural killer (NK) cells eliminate cancer cells. CTL and NK work in parallel, but most studies address them individually. C_LIO_LIIn a new human experimental model, antigen-specific CTL and NK cell cytotoxicity interdependence against melanoma is shown. C_LIO_LIWhereas high numbers of antigen-specific CTL and NK cells eliminate all melanoma cells, lower, more physiological numbers induce resistance, in case secondary CTL or NK cell exposure follow initial CTL cell exposure or if secondary NK cell exposure follows initial NK cell exposure; only if secondary CTL exposure follows initial NK cell exposure no resistance of melanoma but even a slight enhancement of cytotoxicity was observed. C_LIO_LIAlterations in HLA-I expression correlated with resistance to NK cells, while reduction in antigen expression correlated with reduced CTL cytotoxicity. CTL cytotoxicity was rescued beyond control levels by exogenous antigen. C_LIO_LIThe results should help to better understand and optimize immune therapies against cancer. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/150672v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@19fef9eorg.highwire.dtl.DTLVardef@12cae81org.highwire.dtl.DTLVardef@749eeforg.highwire.dtl.DTLVardef@6e7cf1_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2020

High glucose attenuates Ca2+ influx in cytotoxic T lymphocytes upon target recognition

The killing efficiency of cytotoxic T lymphocytes (CTLs) is tightly regulated by intracellular Ca2+ concentration. Glucose is the key energy source for CTLs, lack of which significantly impairs CTL activation, proliferation and effector functions. The impact of high glucose on Ca2+ influx in CTLs remains largely elusive. In this work, we stimulated primary human CD8+ T cells in medium containing either 25 mM (high glucose, HG) or 5.6 mM glucose (normal glucose, NG). We found that store-operated calcium entry (SOCE) induced by thapsigargin (Tg) is elevated in HG-cultured CTLs compared to their counterparts in NG. Unexpectedly, the Ca2+ influx elicited by recognition of target cells is reduced in HG-cultured CTLs. Under HG condition, STIM1 and STIM2, the calcium sensors in the endoplasmic reticulum (ER), were down-regulated; ORAI1, the main structural component of calcium-release activated channels, remained unchanged, whereas ORAI2 and ORAI3 were up-regulated. The fraction of necrosis of HG-cultured CTLs was enhanced after killing without affecting glucose uptake. Thus, our findings reveal that HG has a distinctive impact on Tg-evoked SOCE and target recognition-induced Ca2+ influx in CTLs and causes more CTL death after killing, suggesting a novel regulatory role of high glucose on modulating CTL functions.

immunology↗