Journal: RSC Advances

Article Title: CellTrap: an instrument-free microfluidic platform for cell–cell interactions at stochastically generated effector-to-target ratios

doi: 10.1039/d6ra02345b

Figure Lengend Snippet: Characterization of the CellTrap device with 1024 traps. (A) The experimental loading frequencies ( f ) of individual particles, i.e. , green and red, are compared to the theoretical Poisson distributions ( P ) for λ 1 = 0.51 (green) and λ 2 = 0.54 (red). (B) A combinatorial loading distribution of the two particle types was analyzed using a double Poisson distribution, which is compared with the experimental dual loading frequency. (C) Representative bright-field, fluorescence, and overlay images of red and green particles trapped in a channel at different k 1 : k 2 ratios. (D) The experimental loading frequencies ( f ) and the theoretical Poisson distributions ( P ) are plotted for the U87 GFP and NK92 IL2 cells seeded in the CellTrap device with λ 1 = 0.83 (U87 GFP ) and λ 2 = 0.58 (NK92 IL2 ). (E) The experimental dual loading frequency and theoretical double Poisson distributions result in varying E : T or k 1 : k 2 ratios. (F) Representative bright-field, fluorescence, and overlay images of cancer (purple) and immune (white arrows) cells trapped inside a channel at different E : T ratios. (scale bars: 30 µm).

Article Snippet: The human cell lines NK92 IL2 , U87, K562, and LS174T were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Fluorescence

Journal: RSC Advances

Article Title: CellTrap: an instrument-free microfluidic platform for cell–cell interactions at stochastically generated effector-to-target ratios

doi: 10.1039/d6ra02345b

Figure Lengend Snippet: Response of PBMCs and NK92 IL2 against U87 GFP cells. (A) Fluorescence intensity of U87 GFP cells decreases significantly after 4 h of co-incubation with PBMCs at E : T = ≥1 : ≥1. This data is curated from two independent CellTrap devices ( N = 2), where, in total, 97 traps were analyzed ( n = 97). (B) Inside one of the CellTrap devices used in (A), control traps with only one U87 GFP cell per trap, i.e. , E : T = 0 : 1, were analyzed, maintaining a stable fluorescence signal over 14 h ( N = 1, n = 18). (C) Representative time-lapse images of U87 GFP cells interacting with PBMCs at different E : T ratios, along with the control group containing only U87 GFP cells. (D) Fluorescence intensity of U87 GFP cells decreases significantly after 4 h of co-incubation with NK92 IL2 at E : T = 1 : 1. This data is curated from four independent CellTrap devices ( N = 4), where, in total, 213 traps with E : T = 1 : 1 were analyzed ( n = 213). (E) Inside one of the CellTrap devices used in (D), control traps with only one U87 GFP cell per trap, i.e. , E : T = 0 : 1, were analyzed, maintaining a fluorescence signal over 14 h ( N = 1, n = 50). (F) Representative time-lapse images of U87 GFP cells interacting with NK92 IL2 at different E : T ratios, along with the control group containing only U87 GFP cells. (G) Fluorescence intensity of U87 GFP at 0 h and 14 h of co-incubation with NK92 IL2 at E : T = 1 : 1, 1 : 2, 2 : 1 and 2 : 2. The intensity drop is significant across all E : T ratios except 1 : 2. This data is curated from the same CellTrap devices used in (D).

Article Snippet: The human cell lines NK92 IL2 , U87, K562, and LS174T were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Fluorescence, Incubation, Control

Journal: RSC Advances

Article Title: CellTrap: an instrument-free microfluidic platform for cell–cell interactions at stochastically generated effector-to-target ratios

doi: 10.1039/d6ra02345b

Figure Lengend Snippet: Calcium flux and killing response of immune cells against cancer cells. (A) Calcium flux (normalized intensity) in NK92 IL2 immune cells varies over time in the presence of various cancer cell lines (U87, LS174T, K562). NK92 IL2 cells alone show a flat response (control). Each grey line represents a single immune cell tracked. Representative images show NK92 IL2 cells (green) and cancer cells (U87, LS174T, K562) co-incubated in the CellTrap chip at an E : T ratio of 1 : 1. n = number of traps analyzed. (B) The killing response of NK92 IL2 cells at different E : T ratios (1 : 1, 1 : 2, 2 : 1) against cancer cells (U87, LS174T, K562) is quantified and compared with control groups with E : T ratios of ≥1 : 0 and 0 : ≥1. N = number of CellTrap chips analyzed. n = number of traps analyzed. Representative images show NK92 IL2 cells interacting with cancer cells (U87, LS174T, K562 in blue) with varying E : T ratios at 0 and 14 hours. Red color indicates cell death at 14 hours. Scale bars: 25 µm.

Article Snippet: The human cell lines NK92 IL2 , U87, K562, and LS174T were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Control, Incubation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Aiolos and Eos drive distinct human TH17 functional states

doi: 10.1007/s00018-026-06089-1

Figure Lengend Snippet: Identification of Distinct Human TH17 Cell Subsets and Generation of Stable TH17 Clones from PBMC for Functional Characterization. A Schematic representation of the workflow to generate T H 17-IL22 + /IFNg + and T H 17-IL-10 + clones used to perform bulk ATAC-seq and RNA-seq data sets. In brief, peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood using density gradient centrifugation. The samples were enriched for CD4 + CCR6 + CXCR3- TH17 cells, referred to as “bulk TH17 cells.” Viable IL-17-producing cells were isolated by flow cytometry following a 3-hour stimulation with PMA and ionomycin using a IL-17 capture assay. The single TH17 cell clones were sorted into 384-well plates and expanded with allogeneic γ-irradiated feeder cells and phytohemagglutinin in complete medium containing IL-2. After approximately ten days, clones were transferred to 96-well plates for expansion, and following 2–3 weeks, their cytokine profiles were analyzed. T cell clones were then evaluated at two stages: day 0 (resting state) and day 5 (activated state). On day 5, they were stimulated for 48 hours with anti-CD3 and CD28, followed by an additional 3 days in uncoated plates. On both evaluation days, cells underwent further stimulation — 5 hours for protein analysis and 2 hours for RNA and chromatin-accessibility (ATAC-seq) analysis. Only TH17 clones exhibiting a stable cytokine profile after two rounds of resting and reactivation were selected for RNA-seq and ATAC-seq analysis. B Intracellular staining of IL-17 and IFNγ (top) and IL-22 and IL-10 (bottom) in a T H 17-IL10 + clone (right) and a T H 17-IL22 + /IFNg + clone (left) in the resting state (Day 0) and 5 days post-activation (Day 5). Numbers in quadrants indicate percent cells. C Frequency of IL-17+, IL-10+, IFNγ+, and IL-22+ cells among 6 independent TH17-IL-22 + /IFNγ + (left) and TH17-IL-10 + (right) clones at Day 0 and Day 5. Each symbol represents an individual T cell clone ( n = 6); data are shown as mean ± s.e.m. * P < 0.05, ** P < 0.01 (one-way ANOVA). TH17 clones were selected for RNA and ATAC-seq analysis based on the following criteria: ≥50% IL-17A+ cells at Day 0, ≥15% IL-22+ cells at Day 0 and Day 5, ≥15% IFNγ+ cells at Day 0 and Day 5 for TH17-IL-22 + /IFNγ + clones; ≥50% IL-17A+ cells at Day 0, ≥15% IL-10+ cells at Day 5 for TH17-IL-10 + clones

Article Snippet: IL2 , Hs00174114_m1 , FAM-MGB.

Techniques: Clone Assay, Functional Assay, RNA Sequencing, Isolation, Gradient Centrifugation, Flow Cytometry, Irradiation, Staining, Activation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Aiolos and Eos drive distinct human TH17 functional states

doi: 10.1007/s00018-026-06089-1

Figure Lengend Snippet: Lenalidomide Enhances IL-22/IFNγ and Inhibits IL-10 Independent of IL-2. A Schematic representation of the workflow: human memory TH17 cells were isolated from healthy donors and were activated with anti-CD3 and anti-CD28 for 4 days in presence of blocking antibody against IL-2/IL-2Rβ or recombinant human (rh) IL-2 (100 U/ml or 500 U/ml) in presence of DMSO or lenalidomide (1uM). B Frequency of cytokines in memory TH17 cells 4 days after activation and 4 hours after PMA/ionomycin restimulation. C Relative gene expression of cytokines in memory TH17 cells, 4 days after activation. Each symbol represents an individual memory TH17 donor, n = 6; mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 (one-way ANOVA)

Article Snippet: IL2 , Hs00174114_m1 , FAM-MGB.

Techniques: Isolation, Blocking Assay, Recombinant, Activation Assay, Gene Expression

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Aiolos and Eos drive distinct human TH17 functional states

doi: 10.1007/s00018-026-06089-1

Figure Lengend Snippet: Lenalidomide Promotes Inflammatory Responses in TH17 Cells from Follicular Lymphoma Patients in vivo. A UMAP visualization of 1990 TH17 cells from peripheral blood of follicular lymphoma patients before and after lenalidomide treatment, clustered into two groups: C1 (classic TH17) and C2 (inflammatory TH17). Small UMAP (right) confirms all cells express TH17 signatures. Heatmap shows expression of TH17 signature genes across both clusters, with module scores indicating TH17 signature expression levels. B UMAP projections showing IL-10 and IL-22 clonal signatures at Day 0 (baseline) and Day 7 (post-lenalidomide) treatment. Module scores represent the expression levels of IL-10+ regulatory versus IL-22+ inflammatory gene signatures across the TH17 cell populations. C Volcano plots showing differentially expressed genes between Day 0 (baseline) and Day 7 (post-lenalidomide) treatment for C1 (classic TH17, left) and C2 (inflammatory TH17, right) clusters. Notable upregulated genes include TIMP1, CCL4, IFNG, TNF, and LAG3 in C1, and GZMB, GZMH, GNLY and NKG7 in C2. D Pathway enrichment analysis comparing Day 7 (post-lenalidomide) versus Day 0 (baseline) conditions. Left panel shows hallmark pathways with normalized enrichment scores; right panels show gene set enrichment analysis (GSEA) plots for interferon-γ response, interferon-α response, and IL-2/STAT5 signaling pathways

Article Snippet: IL2 , Hs00174114_m1 , FAM-MGB.

Techniques: In Vivo, Expressing, Protein-Protein interactions