Imaging flow cytometry: A method for examining dynamic native FOXO1 localization in human lymphocytes

Hritzo, Molly; Courneya, Jean-Paul; Golding, Amit

doi:10.1016/j.jim.2018.01.001

Cited by 11 publications

(7 citation statements)

References 36 publications

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“…We used imaging flow cytometry, a technique that combines the advantages of flow cytometry and microscopy and allows the detection and analysis of rare cells within whole cell populations on the basis of their morphological and staining characteristics (Basiji and O'Gorman, 2015;Doan et al, 2018;Hritzo et al, 2018). We thus acquired and analyzed a significant number of relatively rare events of T cell divisions by precisely identifying cells in telophase.…”

Section: Discussionmentioning

confidence: 99%

“…Telophase is the bona fide cell cycle phase where unambiguous measurement of molecular repartition in nascent daughter cells is performed (Chang et al, 2007;Filby et al, 2011). Lytic granule repartition during human CD8 + T cell division was evaluated using imaging flow cytometry, a technique that combines the advantages of both flow cytometry and microscopy (Basiji and O'Gorman, 2015;Doan et al, 2018;Hritzo et al, 2018). This approach allowed us to collect and analyze a substantial number of cells and to visualize and assess the repartition of molecules of interest within individual cells that were unambiguously identified as being in telophase.…”

Section: Imaging Flow Cytometry Reveals Uneven Repartition Of Lytic Mmentioning

confidence: 99%

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Stochastic asymmetric repartition of lytic machinery in dividing CD8+ T cells generates heterogeneous killing behavior

Lafouresse

Jugele

Müller

et al. 2021

eLife

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Cytotoxic immune cells are endowed with a high degree of heterogeneity in their lytic function, but how this heterogeneity is generated is still an open question. We therefore investigated if human CD8+ T cells could segregate their lytic components during telophase, using imaging flow cytometry, confocal microscopy, and live-cell imaging. We show that CD107a+-intracellular vesicles, perforin, and granzyme B unevenly segregate in a constant fraction of telophasic cells during each division round. Mathematical modeling posits that unequal lytic molecule inheritance by daughter cells results from the random distribution of lytic granules on the two sides of the cleavage furrow. Finally, we establish that the level of lytic compartment in individual cytotoxic T lymphocyte (CTL) dictates CTL killing capacity.

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Section: Discussionmentioning

confidence: 99%

Section: Imaging Flow Cytometry Reveals Uneven Repartition Of Lytic Mmentioning

confidence: 99%

Stochastic asymmetric repartition of lytic machinery in dividing CD8+ T cells generates heterogeneous killing behavior

Lafouresse

Jugele

Müller

et al. 2021

eLife

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“…In contrast to HuT78 cell line, HuT102 cell line is known to expand slowly with varied degrees of overall viability (34). Nevertheless, the effects of different inhibitors on these leukemic cell line's viability and proliferation have been extensively evaluated in vitro to identify the novel therapeutic agents for patients with cutaneous T cell lymphomas (35)(36)(37)(38).…”

Section: Exogenous Sema4a Promotes Hut Cell Survival and Stabilizes Tmentioning

confidence: 99%

Semaphorin 4A Stabilizes Human Regulatory T Cell Phenotype via Plexin B1

Chapoval

Hritzo

et al. 2019

ImmunoHorizons

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We previously reported that neuroimmune semaphorin (Sema) 4A regulates the severity of experimental allergic asthma and increases regulatory T (Treg) cell numbers in vivo; however, the mechanisms of Sema4A action remain unknown. It was also reported that Sema4A controls murine Treg cell function and survival acting through neuropilin 1 (NRP-1) receptor. To clarify Sema4A action on human T cells, we employed T cell lines (HuT78 and HuT102), human PBMCs, and CD4 + T cells in phenotypic and functional assays. We found that HuT78 demonstrated a T effector-like phenotype (CD4 + CD25 low Foxp3 2), whereas HuT102 expressed a Treg-like phenotype (CD4 + CD25 hi Foxp3 +). Neither cell line expressed NRP-1. HuT102 cells expressed Sema4A counter receptor Plexin B1, whereas HuT78 cells were Sema4A +. All human peripheral blood CD4 + T cells, including Treg cells, expressed PlexinB1 and lacked both NRP-1 and-2. However, NRP-1 and Sema4A were detected on CD3 negative CD4 intermediate human monocytes. Culture of HuT cells with soluble Sema4A led to an upregulation of CD25 and Foxp3 markers on HuT102 cells. Addition of Sema4A increased the relative numbers of CD4 + CD25 + Foxp3 + cells in PBMCs and CD4 + T cells, which were NRP-1 negative but PlexinB1 + , suggesting the role of this receptor in Treg cell stability. The inclusion of anti-PlexinB1 blocking Ab in cultures before recombinant Sema4A addition significantly decreased Treg cell numbers as compared with cultures with recombinant Sema4A alone. Sema4A was as effective as TGF-b in inducible Treg cell induction from CD4 + CD25 depleted cells but did not enhance Treg cell suppressive activity in vitro. These results suggest strategies for the development of new Sema4A-based therapeutic measures to combat allergic inflammatory diseases. ImmunoHorizons, 2019, 3: 71-87.

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“… 1–3 FOXO1 is a transcription factor involved at critical early and late B cell development checkpoints; however, its role in regulating peripheral B cell tolerance is not fully understood. We have applied our published approach for using imaging flow cytometry (IFC) 4 to study native FOXO1 localisation in human lymphocytes to peripheral blood samples from healthy individuals versus patients with SLE. We report, here, on dramatic cytoplasmic localisation of FOXO1 in two peripheral B cell SLE subsets: IgD-CD27+ (switched memory) B cells and IgD-CD27- (atypical memory) B cells.…”

Section: Introductionmentioning

confidence: 99%

Cytoplasmic FOXO1 identifies a novel disease-activity associated B cell phenotype in SLE

Ahye

Golding

2018

Lupus Sci Med

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Systemic lupus erythematosus (SLE) is a manifestation of hyperactivated lymphocytes and results, in part, from the loss of normal tolerance checkpoints. FOXO1 is a transcription factor involved at critical early and late B cell development checkpoints; however, its role in regulating peripheral B cell tolerance is not fully understood. We have applied our published approach for using imaging flow cytometry to study native FOXO1 localisation in human lymphocytes to peripheral blood samples from healthy individuals versus patients with SLE. We report, here, on dramatic cytoplasmic localisation of FOXO1 in two peripheral B cell SLE subsets: IgD-CD27+ (class-switched memory) B cells and IgD-CD27- (atypical memory) B cells. The latter, so-called ‘Double Negative’ (DN) B cells have previously been shown to be increased in SLE and enriched in autoreactive clones. Cytoplasmic-predominant FOXO1 (CytoFOX) B cells are significantly increased in patients with SLE as compared to healthy controls, and the levels of CytoFoOX DN B cells correlate directly with SLE disease activity. The highest abundance of CytoFox DN B cells was observed in African American females with SLE Disease Activity Index (SLEDAI)≥6. The phenotype of CytoFOX DN B cells in SLE includes uniquely low CD20 expression and high granularity/side scatter. As FOXO1 phosphorylation downstream of B cell receptor-dependent signalling is required for nuclear exclusion, CytoFOX B cells likely represent a high state of B cell activation with excess signalling and/or loss of phosphatase activity. We hypothesise that CytoFOX B cells in lupus represent a novel biomarker for the expansion of pathological, autoreactive B cells which may provide new insights into the pathophysiology of SLE.

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Imaging flow cytometry: A method for examining dynamic native FOXO1 localization in human lymphocytes

Cited by 11 publications

References 36 publications

Stochastic asymmetric repartition of lytic machinery in dividing CD8+ T cells generates heterogeneous killing behavior

Stochastic asymmetric repartition of lytic machinery in dividing CD8+ T cells generates heterogeneous killing behavior

Semaphorin 4A Stabilizes Human Regulatory T Cell Phenotype via Plexin B1

Cytoplasmic FOXO1 identifies a novel disease-activity associated B cell phenotype in SLE

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