Qualitative and quantitative analysis of PMN/T-cell interactions by InFlow and super-resolution microscopy

Balta, Emre; Stopp, Julian; Castelletti, Laura; Kirchgessner, h.c. mult. M.; Samstag, Yvonne; Wabnitz, Guido H.

doi:10.1016/j.ymeth.2016.09.013

Cited by 13 publications

(13 citation statements)

References 49 publications

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“…In addition to this intra-lineage plasticity, repair-associated neutrophils are capable of trans-lineage plasticity (i.e., plasticity by transdifferentiation) (45, 46). Challenging the classic view of neutrophils as terminally differentiated leukocytes fully committed to phagocytosis, new evidence shows that neutrophils can acquire phenotypic and functional properties typically associated with professional antigen-presenting cells (APCs) (47, 48)—e.g., MHC II expression and co-stimulatory molecules (49).…”

Section: Plasticity Of Neutrophils and Macrophages In Repairmentioning

confidence: 99%

Immune-Mediated Repair: A Matter of Plasticity

et al. 2017

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Though the immune system is generally defined as a system of defense, it is increasingly recognized that the immune system also plays a crucial role in tissue repair and its potential dysregulations. In this review, we explore how distinct immune cell types are involved in tissue repair and how they interact in a process that is tightly regulated both spatially and temporally. We insist on the concept of immune cell plasticity which, in recent years, has proved fundamental for the success/understanding of the repair process. Overall, the perspective presented here suggests that the immune system plays a central role in the physiological robustness of the organism, and that cell plasticity contributes to the realization of this robustness.

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Section: Plasticity Of Neutrophils and Macrophages In Repairmentioning

confidence: 99%

Immune-Mediated Repair: A Matter of Plasticity

et al. 2017

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“…The fluorescence intensities of the GrA staining were measured in both masks and a ratio was calculated to achieve the percentage of GrA in the contact zone. For quantitative evaluation we set a threshold, so that CTLs with an GrA enrichment in the cytolytic immune synapse (IS) of at least 30% were counted as cells with “GrA in immune synapse (IS).” This percentage was chosen based on preceding manual evaluation and judgments (see also [Balta et al, ]). Interestingly, GrA, was already present in the contact zone in 22% of the CTLs, when target cells were left unloaded (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The gating strategy was performed as described in the results part by applying the following masks: “Area_Morphology(M02, Ch02)” for calculating nuclear area, “MC And Not Morphology(M05_Ch05)” for defining target cells; “Threshold(Mo5_Ch05, 60)” as T‐cell mask; “Threshold(Mo5_Ch05, 60) And Valley(M02_Ch02,3)” were used as Synapse mask. For a detailed description of masks see [Balta et al, ; Wabnitz et al, ; Wabnitz and Samstag, ].…”

Section: Methodsmentioning

confidence: 99%

Imaging Flow Cytometry for Multiparametric Analysis of Molecular Mechanism Involved in the Cytotoxicity of Human CD8⁺ T-cells

2017

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The clearance of tumors or virus infected cells is a crucial task of the immune system. Cytotoxic T-cells (CTLs) are able to detect and to kill such altered host cells. Given the recent success of checkpoint inhibitors for tumor therapy, it becomes more and more important to understand the biology of T-cell mediated target cell killing. Tests that allow analyzing the biology of CTLs are either based on flow cytometry or fluorescence microscopy. Thus, they either lack image-based information or have a poor statistical robustness. Therefore, we describe an approach to quantify CTL-mediated cytotoxicity using imaging flow cytometry. Using activated primary human cytotoxic T-cells as CTLs and P815 as target cells, we show that both the evaluation of target cell death and the biology of CTLs can be evaluated in parallel. This enables to gain information about CTL-mediated cytotoxicity in samples from patients important for translational medicine. J. Cell. Biochem. 118: 2528-2533, 2017. © 2017 Wiley Periodicals, Inc.

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“…Imaging flow cytometry (hereafter termed ImageStream) was performed as described ( 20 ). Cells were analyzed by ImageStream (Amnis, Merck Millipore, Darmstadt, Germany).…”

Section: Methodsmentioning

confidence: 99%

Interaction and Mutual Activation of Different Innate Immune Cells Is Necessary to Kill and Clear Hepatitis C Virus-Infected Cells

et al. 2017

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Innate immune cells can sense hepatitis C virus (HCV)-infected cells and respond with anti-viral actions including secretion of interferons (IFNs). In previous studies, the response of individual innate immune cells against HCV was analyzed in detail. We hypothesized that interaction of multiple innate immune cells increases the magnitude of the immune response and eventually leads to clearance of HCV-infected cells. To investigate this, we co-cultured Huh-7 HCV subgenomic replicon (SGR) cells with peripheral blood mononuclear cells (PBMCs). We confirm secretion of IFNα by plasmacytoid dendritic cells (pDCs) and IFNγ by natural killer (NK) cells in the co-culture setup. Moreover, we observed that also monocytes contribute to the anti-viral response. Flow cytometry and ImageStream analysis demonstrated that monocytes take up material from HCV SGR cells in co-culture with PBMCs. Preceding the uptake, PBMCs caused apoptosis of HCV SGR cells by tumor necrosis factor-related apoptosis inducing ligand (TRAIL) expression on NK cells. We observed that only the interplay of monocytes, pDCs, and NK cells resulted in efficient clearance of HCV SGR cells, while these cell populations alone did not kill HCV SGR cells. Despite similar TRAIL receptor expression on Huh-7 control cells and HCV SGR cells, HCV activated PBMCs specifically killed HCV SGR cells and did not target Huh-7 control cells. Finally, we showed that HCV replicating cells per se are sensitive toward TRAIL-induced apoptosis. Our results highlight the importance of the interplay of different innate immune cells to initiate an efficient, rapid, and specific response against HCV-infected cells.

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Qualitative and quantitative analysis of PMN/T-cell interactions by InFlow and super-resolution microscopy

Cited by 13 publications

References 49 publications

Immune-Mediated Repair: A Matter of Plasticity

Immune-Mediated Repair: A Matter of Plasticity

Imaging Flow Cytometry for Multiparametric Analysis of Molecular Mechanism Involved in the Cytotoxicity of Human CD8⁺ T-cells

Interaction and Mutual Activation of Different Innate Immune Cells Is Necessary to Kill and Clear Hepatitis C Virus-Infected Cells

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Qualitative and quantitative analysis of PMN/T-cell interactions by InFlow and super-resolution microscopy

Cited by 13 publications

References 49 publications

Immune-Mediated Repair: A Matter of Plasticity

Immune-Mediated Repair: A Matter of Plasticity

Imaging Flow Cytometry for Multiparametric Analysis of Molecular Mechanism Involved in the Cytotoxicity of Human CD8+ T-cells

Interaction and Mutual Activation of Different Innate Immune Cells Is Necessary to Kill and Clear Hepatitis C Virus-Infected Cells

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Imaging Flow Cytometry for Multiparametric Analysis of Molecular Mechanism Involved in the Cytotoxicity of Human CD8⁺ T-cells