light-sheet-microscopy-for-three-dimensional-visualization-human

Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or ...

Saarland University

Ca microdomains and spatially resolved Ca signals are highly relevant for cell function. In T cells, local Ca signaling at the immunological synapse (IS) is required for downstream effector functions. We present experimental evidence that the relocation of the MTOC towards the IS during polarization drags mitochondria along with the microtubule network. From time-lapse fluorescence microscopy we conclude that mitochondria rotate together with the cytoskeleton towards the IS. We hypothesize that this movement of mitochondria towards the IS together with their functionality of absorption and spatial redistribution of Ca is sufficient to significantly increase the cytosolic Ca concentration. To test this hypothesis we developed a whole cell model for Ca homoeostasis involving specific geometries for mitochondria and use the model to calculate the spatial distribution of Ca concentrations within the cell body as a function of the rotation angle and the distance from the IS. We find that an inhomogeneous distribution of PMCA pumps on the cell membrane, in particular an accumulation of PMCA at the IS, increases the global Ca concentration and decreases the local Ca concentration at the IS with decreasing distance of the MTOC from the IS. Unexpectedly, a change of CRAC/Orai activity is not required to explain the observed Ca changes. We conclude that rotation-driven relocation of the MTOC towards the IS together with an accumulation of PMCA pumps at the IS are sufficient to control the observed Ca dynamics in T-cells during polarization.

Cytoskeleton rotation relocates mitochondria to the immunological synapse and increases calcium signals.

Natural killer (NK) cells play a central role during innate immune responses by eliminating pathogen-infected or tumorigenic cells. In the microenvironment, NK cells encounter not only target cells but also other cell types including non-target bystander cells. The impact of bystander cells on NK killing efficiency is, however, still elusive. In this study we show that the presence of bystander cells, such as P815, monocytes or HUVEC, enhances NK killing efficiency. With bystander cells present, the velocity and persistence of NK cells were increased, whereas the degranulation of lytic granules remained unchanged. Bystander cell-derived HO was found to mediate the acceleration of NK cell migration. Using mathematical diffusion models, we confirm that local acceleration of NK cells in the vicinity of bystander cells reduces their search time to locate target cells. In addition, we found that integrin β chains (β1, β2 and β7) on NK cells are required for bystander-enhanced NK migration persistence. In conclusion, we show that acceleration of NK cell migration in the vicinity of HO-producing bystander cells reduces target cell search time and enhances NK killing efficiency.

Bystander cells enhance NK cytotoxic efficiency by reducing search time.

The actin-binding protein profilin1 (PFN1) plays a central role in actin dynamics, which is essential for cytotoxic T lymphocyte (CTL) functions. The functional role of PFN1 in CTLs, however still remains elusive. Here, we identify PFN1 as the only member of the profilin family expressed in primary human CD8 T cells. Using in vitro assays, we find that PFN1 is a negative regulator of CTL-mediated elimination of target cells. Furthermore, PFN1 is involved in activation-induced lytic granule (LG) release, CTL migration and modulation of actin structures at the immunological synapse (IS). During CTL migration, PFN1 modulates the velocity, protrusion formation patterns and protrusion sustainability. In contrast, PFN1 does not significantly affect migration persistence and the rates of protrusion emergence and retraction. Under in vitro conditions mimicking a tumor microenvironment, we show that PFN1 downregulation promotes CTL invasion into a 3D matrix, without affecting the viability of CTLs in a hydrogen peroxide-enriched microenvironment. Highlighting its potential relevance in cancer, we find that in pancreatic cancer patients, PFN1 expression is substantially decreased in peripheral CD8 T cells. Taken together, we conclude that PFN1 is a negative regulator for CTL-mediated cytotoxicity and may have an impact on CTL functionality in a tumor-related context.

Human profilin 1 is a negative regulator of CTL mediated cell-killing and migration.

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are required to eliminate cancer cells. We analysed the Ca dependence of CTL and NK cell cytotoxicity and found that in particular CTLs have a very low optimum of [Ca ] (between 122 and 334 nm) and [Ca ] (between 23 and 625 μm) for efficient cancer cell elimination, well below blood plasma Ca levels. As predicted from these results, partial down-regulation of the Ca channel Orai1 in CTLs paradoxically increases perforin-dependent cancer cell killing. Lytic granule release at the immune synapse between CTLs and cancer cells has a Ca optimum compatible with this low Ca optimum for efficient cancer cell killing, whereas the Ca optimum for CTL migration is slightly higher and proliferation increases monotonously with increasing [Ca ] . We propose that a partial inhibition of Ca signals by specific Orai1 blockers at submaximal concentrations could contribute to tumour elimination.

Article	Year
Cytoskeleton rotation relocates mitochondria to the immunological synapse and increases calcium signals. Cell calcium\| PubMed ID: 27451384	2016
Bystander cells enhance NK cytotoxic efficiency by reducing search time. Scientific reports\| PubMed ID: 28287155	2017
Human profilin 1 is a negative regulator of CTL mediated cell-killing and migration. European journal of immunology\| PubMed ID: 28688208	2017
A calcium optimum for cytotoxic T lymphocyte and natural killer cell cytotoxicity. The Journal of physiology\| PubMed ID: 29368348	2018

Renping Zhao

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