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 ...

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 are effector CD8 T cells that eradicate infected and malignant cells. Here we show that the transcription factor NFATc1 controls the cytotoxicity of mouse cytotoxic T lymphocytes. Activation of Nfatc1  cytotoxic T lymphocytes showed a defective cytoskeleton organization and recruitment of cytosolic organelles to immunological synapses. These cells have reduced cytotoxicity against tumor cells, and mice with NFATc1-deficient T cells are defective in controlling Listeria infection. Transcriptome analysis shows diminished RNA levels of numerous genes in Nfatc1  CD8 T cells, including Tbx21, Gzmb and genes encoding cytokines and chemokines, and genes controlling glycolysis. Nfatc1  , but not Nfatc2  CD8 T cells have an impaired metabolic switch to glycolysis, which can be restored by IL-2. Genome-wide ChIP-seq shows that NFATc1 binds many genes that control cytotoxic T lymphocyte activity. Together these data indicate that NFATc1 is an important regulator of cytotoxic T lymphocyte effector functions.NFAT nuclear translocation has been shown to be required for CD8 T cell cytokine production in response to viral infection. Here the authors show NFATc1 controls the cytotoxicity and metabolic switching of activated CD8 T cells required for optimal response to bacteria and tumor cells.

NFATc1 controls the cytotoxicity of CD8 T cells.

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
Human profilin 1 is a negative regulator of CTL mediated cell-killing and migration. European journal of immunology\| PubMed ID: 28688208	2017
NFATc1 controls the cytotoxicity of CD8 T cells. Nature communications\| PubMed ID: 28894104	2017
A calcium optimum for cytotoxic T lymphocyte and natural killer cell cytotoxicity. The Journal of physiology\| PubMed ID: 29368348	2018

Rouven Schoppmeyer

Biography

JoVE Journal Publications

Other Publications