Journal: Kidney International Reports

Article Title: Early-Stage B-cells Predict Relapse After Rituximab Treatment in Patients With Membranous Nephropathy

doi: 10.1016/j.ekir.2026.106365

Figure Lengend Snippet: Reconstitution of B-cells over time. To characterize B-cell subsets, peripheral blood mononuclear cells were stained with fluorochrome-conjugated monoclonal antibodies (BD Biosciences) directed against the following antigens: CD45 (V500-C), CD3 (APC), CD19 (APC-H7), CD27 (BV421), IgD (PE), CD38 (BV711), CD4 (BV605), and CD8 (PE), as well as 7-AAD (Miltenyi Biotech). The stained cells were then analyzed using multicolor flow cytometry (BD FACS Lyric). The subsets of gated CD19 + cells were identified based on surface marker expression as follows: naïve (CD19+CD27-IgD+), nonswitched memory (CD19+CD27+IgD+), switched memory (CD19+CD27+IgD-), and double negative (CD19+CD27-IgD-); and within the CD19+CD38++ population, we distinguished transitional cells (CD19+CD38++CD27-IgD+), plasmablasts (CD19+CD38++CD27+IgD-), and double-negative CD38 + cells (CD19+CD38++CD27-IgD-). B-cell subsets were expressed as a percentage of the total lymphocyte count. (a) B-cell subpopulations were assessed in 39 patients at baseline and in 36 patients at 3 (M3), 6 (M6), 9 (M9), 12 (M12), and 18 (M18) months following the first rituximab infusion. The 3 patients who did not receive treatment were excluded from the follow-up. Complete depletion of B-cells was observed in all patients at M3 post-rituximab for all B-cell subpopulations. CD19 + cells reappeared 6 months after rituximab infusion. Naive cells re-emerged the most among B-cells, followed by CD38 + cells, transitional cells and finally memory cells. Data are shown as mean values (dots). (b–k) B-cell subpopulations at baseline and at subsequent time points were compared between relapsing patients ( n = 8) and nonrelapsing patients ( n = 19); the 2 patients who received additional anti-CD20 infusions were excluded from subsequent analyses. Data are shown as medians and interquartile range (IQR). P -values were calculated by comparing the median values of each cell subpopulation between relapsing and nonrelapsing patients using a nonparametric, unpaired Mann–Whitney U test.

Article Snippet: To characterize B-cell subsets, peripheral blood mononuclear cells were stained with fluorochrome-conjugated monoclonal antibodies (BD Biosciences) directed against the following antigens: CD45 (V500-C), CD3 (APC), CD19 (APC-H7), CD27 (BV421), IgD (PE), CD38 (BV711), CD4 (BV605), and CD8 (PE), as well as 7-AAD (Miltenyi Biotech).

Techniques: Staining, Bioprocessing, Flow Cytometry, Marker, Expressing, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Distinct dendritic cell cytoskeletal programs dictate synapse architecture and CD8 + T cell fate

doi: 10.3389/fimmu.2026.1716644

Figure Lengend Snippet: Dendritic cells form distinct synapse morphologies dictated by CD70 signaling. (A) Schematic of the pseudo-synapse model using total internal reflection fluorescence (TIRF) microscopy. (B) Representative TIRF images BMDCs forming either pancake or firework synapses on anti-MHCI-coated glass, stained for F-actin and quantification of the prevalence of each phenotype (n=4 biological replicates, n 1 = 127 cells, n 2 = 155 cells, n 3 = 24 cells, n 4 = 168 cells). (C) Time-lapse TIRF imaging of a live BMDC stained with CellTracker Deep Red, showing the formation and stability of either pancake or firework synapse. Image acquisition was initiated immediately after the stained BMDCs were added to the imaging chambers. Images were acquired every minute for 3 hours at 37 °C in a 5% CO 2 atmosphere. Time stamp is hh:mm. (D) Representative TIRF images showing CD70 and actin expression in pancake vs. firework synapses on MHCI-coated glass. (E) Quantification of membrane-proximal CD70 mean fluorescence intensity (MFI) at the synapse interface (measured by TIRF microscopy) in each synapse phenotype on glass coated with anti-MHCI (aMHCI) alone or with CD27. MFI is normalized to the background (n 1 = 117 cells, n 2 = 109 cells, n 3 = 104 cells). (F) Representative confocal images with orthogonal side-views (G) showing CD70 localization in BMDCs. The arrow indicates CD70 recruitment to the synapse upon CD27 engagement. (G) Quantification of CD70 recruitment to the synapse measured measure by confocal microscopy. Briefly, CD70 mean fluorescence intensity (MFI) was quantified at the synapse and normalized by the CD70 MFI in the whole cell. Cells were classified by their synapse morphologies, namely fireworks or pancakes (n 1 = 41 cells, n 2 = 44 cells, n 3 = 39 cells). (H) Quantification of CD70 recruitment to the synapse measured measure by confocal microscopy. Briefly, CD70 mean fluorescence intensity (MFI) was quantified at the synapse and normalized by the CD70 MFI in the whole cell. Cells were classified by their total CD70 expression. To define CD70 high and CD70 low , the median fluorescence intensity was calculated across all cells pooled from three independent experiments, with cells above the median classified as CD70 high and cells below as CD70 low (n 1 = 41 cells, n 2 = 44 cells, n 3 = 39 cells). (I) Representative confocal images showing CD70 distribution in BMDCs. Images are representative of 3 independent experiments. (J) Frequency of firework synapses in BMDCs, with or without pre-treatment with anti-CD40 (aCD40), on surfaces coated with aMHCI alone or with CD27 (n 1 = 547 cells, n 2 = 629 cells, n 3 = 1028 cells). Data are shown as mean ± SD (n=3 biological replicates). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.00; ns, non-significant. Main scale bars: 20 µm, inlets scale bars = 10µm. Statistical significance was determined using two-way ANOVA. Note: These images illustrate the two distinct morphological states initially observed in LPS-matured BMDCs. The dendritic cell identity and subpopulation characteristics corresponding to these morphologies are validated by marker analysis (including Zbtb46) in Figures 2D, E .

Article Snippet: Cover Glass 18 mm #1.5 (Marinefeld, 0117580) was functionalized by coating with anti-MHCI antibody (Invitrogen, 16-5999-82) and CD27 recombinant protein (R&D Systems, 574-CD-050) at a concentration of 10 μg/mL in sterile PBS.

Techniques: Fluorescence, Microscopy, Staining, Imaging, Expressing, Membrane, Confocal Microscopy, Marker