31 research outputs found
Plasma Membrane Calcium ATPase Regulates Stoichiometry of CD4+ T-Cell Compartments
Immune responses involve mobilization of T cells within naĂŻve and memory compartments.
Tightly regulated Ca2+ levels are essential for balanced immune outcomes. How Ca2+
contributes to regulating compartment stoichiometry is unknown. Here, we show that
plasma membrane Ca2+ ATPase 4 (PMCA4) is differentially expressed in human CD4+ T
compartments yielding distinct store operated Ca2+ entry (SOCE) profiles. Modulation of
PMCA4 yielded a more prominent increase of SOCE in memory than in naĂŻve CD4+ T cell.
Interestingly, downregulation of PMCA4 reduced the effector compartment fraction and
led to accumulation of cells in the naĂŻve compartment. In silico analysis and chromatin
immunoprecipitation point towards Ying Yang 1 (YY1) as a transcription factor regulating
PMCA4 expression. Analyses of PMCA and YY1 expression patterns following activation
and of PMCA promoter activity following downregulation of YY1 highlight repressive role of
YY1 on PMCA expression. Our findings show that PMCA4 adapts Ca2+ levels to cellular
requirements during effector and quiescent phases and thereby represent a potential
target to intervene with the outcome of the immune response
Supra-Molecular Assemblies of ORAI1 at Rest Precede Local Accumulation into Puncta after Activation
The Ca2+ selective channel ORAI1 and endoplasmic reticulum (ER)-resident STIM proteins
form the core of the channel complex mediating store operated Ca2+ entry (SOCE). Using liquid phase
electron microscopy (LPEM), the distribution of ORAI1 proteins was examined at rest and after SOCEactivation at nanoscale resolution. The analysis of over seven hundred thousand ORAI1 positions
revealed a number of ORAI1 channels had formed STIM-independent distinct supra-molecular
clusters. Upon SOCE activation and in the presence of STIM proteins, a fraction of ORAI1 assembled
in micron-sized two-dimensional structures, such as the known puncta at the ER plasma membrane
contact zones, but also in divergent structures such as strands, and ring-like shapes. Our results thus
question the hypothesis that stochastically migrating single ORAI1 channels are trapped at regions
containing activated STIM, and we propose instead that supra-molecular ORAI1 clusters fulfill an
amplifying function for creating dense ORAI1 accumulations upon SOCE-activation
Cytotoxic Efficiency of Human CD8+ T Cell Memory Subtypes
Immunological memory is important 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 well defined, single TEM and TCM cell cytotoxicity has not been quantified.
To quantify cytotoxic efficiency of TEM and TCM, we developed a FRET-based single cell
fluorescent assay with NALM6 target cells which allows analysis of target cell apoptosis,
secondary necrosis following apoptosis, and primary necrosis after TEM- or TCM-target cell
contact. Both, single cell and population cytotoxicity assays reveal a higher cytotoxic
efficiency of TEM compared to TCM, as quantified by target cell apoptosis and secondary
necrosis. Perforin, granzyme B, FasL, but not TRAIL expression are higher in TEM
compared to TCM. Higher perforin levels (likely in combination with higher granzyme
levels) mediate higher cytotoxic efficiency of TEM compared to TCM. Both, TEM and TCM
need the same time to find their targets, however contact time between CTL and target,
time to induce apoptosis, and time to induce secondary necrosis are all shorter for TEM. In
addition, immune synapse formation in TEM appears to be slightly more efficient than in
TCM. Defining and quantifying single TEM and TCM cytotoxicity and the respective
mechanisms is important to optimize future subset-based immune therapies
Recommended from our members
High glucose distinctively regulates Ca2+ influx in cytotoxic T lymphocytes upon target recognition and thapsigargin stimulation
In CTLs: High glucoseâculture enhances thapsigarginâinduced SOCE but decreases target recognitionâinduced Ca2+ influx.
High glucoseâculture regulates expression of ORAIs and STIMs without affecting glucose uptake.
More high glucoseâcultured CTLs are prone to necrosis after execution of killing. (...
Acute Downregulation but Not Genetic Ablation of Murine MCU Impairs Suppressive Capacity of Regulatory CD4 T Cells
By virtue of mitochondrial control of energy production, reactive oxygen species (ROS)
generation, and maintenance of Ca2+ homeostasis, mitochondria play an essential role in modulating
T cell function. The mitochondrial Ca2+ uniporter (MCU) is the pore-forming unit in the main protein
complex mediating mitochondrial Ca2+ uptake. Recently, MCU has been shown to modulate Ca2+
signals at subcellular organellar interfaces, thus fine-tuning NFAT translocation and T cell activation.
The mechanisms underlying this modulation and whether MCU has additional T cell subpopulationspecific effects remain elusive. However, mice with germline or tissue-specific ablation of Mcu did
not show impaired T cell responses in vitro or in vivo, indicating that âchronicâ loss of MCU can
be functionally compensated in lymphocytes. The current work aimed to specifically investigate
whether and how MCU influences the suppressive potential of regulatory CD4 T cells (Treg). We show
that, in contrast to genetic ablation, acute siRNA-mediated downregulation of Mcu in murine Tregs
results in a significant reduction both in mitochondrial Ca2+ uptake and in the suppressive capacity
of Tregs, while the ratios of Treg subpopulations and the expression of hallmark transcription factors
were not affected. These findings suggest that permanent genetic inactivation of MCU may result in
compensatory adaptive mechanisms, masking the effects on the suppressive capacity of Tregs
Modulation of intracellular calcium signaling by microRNA-34a-5p
Adjusting intracellular calcium signaling is an important feature in the regulation of immune cell function and survival. Here we show that miR-34a-5p, a small non-coding RNA that is deregulated in many common diseases, is a regulator of store-operated Ca2+ entry (SOCE) and calcineurin signaling. Upon miR-34a-5p overexpression, we observed both a decreased depletion of ER calcium content and a decreased Ca2+ influx through Ca2+ release-activated Ca2+ channels. Based on an in silico target prediction we identified multiple miR-34a-5p target genes within both pathways that are implicated in the balance between T-cell activation and apoptosis including ITPR2, CAMLG, STIM1, ORAI3, RCAN1, PPP3R1, and NFATC4. Functional analysis revealed a decrease in Ca2+ activated calcineurin pathway activity measured by a reduced IL-2 secretion due to miR-34a-5p overexpression. Impacting SOCE and/or downstream calcineurin/NFAT signaling by miR-34a-5p offers a possible future approach to manipulate immune cells for clinical interventions
TREM2 Is Associated with Advanced Stages and Inferior Prognosis in Oral Squamous Cell Carcinoma
Triggering receptor expressed on myeloid cells 2 (TREM2) is suggested to hamper antitumor immune response in multiple cancers. However, the role of TREM2 in oral squamous cell carcinoma (OSCC) and its expression in tumor-associated macrophages (TAMs) are unknown. In this study, TREM2 expression was analyzed in the primary tumors and corresponding lymph-node metastases of OSCC patients via immunohistochemistry on tissue microarrays. Human peripheral blood mononuclear cells (PBMCs) and single-cell suspensions of tumor and healthy adjacent tissues were analyzed for the presence of TREM2+ macrophages and TAMs using flow cytometry. The serum levels of soluble TREM2 (sTREM2) were quantified using an enzyme-linked immunosorbent assay. High TREM2 expression was associated with advanced UICC stages (Spearmanâs rank correlation (SRC), p = 0.04) and significantly reduced survival rates in primary tumors (multivariate Cox regression, progression-free survival: hazard ratio (HR) of 2.548, 95% confidence interval (CI) of 1.089â5.964, p = 0.028; overall survival: HR of 2.17, 95% CI of 1.021â4.613, p = 0.044). TREM2 expression was significantly increased in the PBMCs of OSCC patients in UICC stage IV compared with healthy controls (ANOVA, p < 0.05). The serum levels of sTREM2 were higher in advanced UICC stages, but they narrowly missed significance (SRC, p = 0.059). We demonstrated that TREM2 was multi-factorially associated with advanced stages and inferior prognosis in OSCC patients and that it could serve as a prognostic biomarker in OSCC patients. Targeting TREM2 has the potential to reshape the local and systemic immune landscape for the potential enhancement of patientsâ prognosis
IDO1 is highly expressed in macrophages of patients in advanced tumour stages of oral squamous cell carcinoma
Purpose Strategies for Indolamine-2,3-dioxygenase 1 (IDO1) inhibition in cancer immunotherapy once produced encouraging results, but failed in clinical trials. Recent evidence indicates that immune cells in the tumour microenvironment,
especially macrophages, contribute to immune dysregulation and therefore might play a critical role in drug resistance.
Methods In this study, we investigated the signifcance of IDO1 expressing immune cells in primary tumours and corresponding lymph node metastases (LNMs) in oral squamous cell carcinoma (OSCC) by immunohistochemistry. The link
between IDO1 and macrophages was investigated by fow cytometry in tumour tissue, healthy adjacent tissue and peripheral
blood mononuclear cells (PBMCs). IDO1 activity (measured as Kynurenine/Tryptophan ratio) was assessed by ELISAs.
Results High IDO1 expression in tumour-infltrating immune cells was signifcantly correlated with advanced stages [Spearmanâs rank correlation (SRC), p=0.027] and reduced progression-free survival (multivariate Cox regression, p=0.034).
IDO1 was signifcantly higher expressed in PBMCs of patients in advanced stages than in healthy controls (ANOVA, p<0.05)
and IDO1+ macrophages were more abundant in intratumoural areas than peritumoural (t test, p<0.001). IDO1 expression
in PBMCs was signifcantly correlated with IDO1 activity in serum (SRC, p<0.05). IDO1 activity was signifcantly higher
in patients with LNMs (t test, p<0.01).
Conclusion All in all, IDO1 expressing immune cells, especially macrophages, are more abundant in advanced stages of
OSCC and are associated with reduced progression-free survival. Further investigations are needed to explore their role
in local and systemic immune response. The IDO1 activity might be a suitable biomarker of metastasis in OSCC patients