Journal: Molecular Therapy. Nucleic Acids

Article Title: Engineered mRNA backbones for gene expression in human T cells

doi: 10.1016/j.omtn.2026.102913

Figure Lengend Snippet: T cell-specific UTRs for optimized CAR expression, reactivity, and tonic signaling (A). Representative flow cytometry analysis of PBMC-derived T cells electroporated with CD19-CAR-encoding constructs using various 5′ UTRs. (B) Quantification of percentages of CD19-CAR + cells in (A). Data are normalized to the HBA1 UTR condition and presented as mean ± SEM from four independent experiments ( n = 4). Kruskal-Wallis test revealed no statistically significant difference ( p = 0.29). (C) MFI of CD19-CAR + cells in (A) normalized to the HBA1 UTR. Data represent four independent experiments ( n = 4). Kruskal-Wallis test indicated a significant difference among groups ( p < 0.005); Dunn’s post hoc test revealed significant reduction in the TNF-UTR group compared to HBA1 ( p = 0.0022). (D) Secreted IFN-γ levels in co-culture supernatants of PBMC-derived T cells electroporated with CD19-CAR mRNA using different 5′ UTRs and CD19 + NALM6 target cells. Data from two healthy donors (D29 and D40) are shown at various effector-to-target (E:T) ratios. (E) Left: interferon gamma ELISA in media taken from co-cultures of PBMC-derived T cells electroporated with CD19-CAR mRNA using different UTRs, either together with CD19 + (filled) or CD19 − (NALM6 KO, empty) at an E:T ratio of 4:1. Right: delta of interferon gamma secretion of E between the co-culture of electroporated T cells with CD19 + vs. CD19 − NALM6 cells. Bars represent mean ± SEM, n = 5 (CD19 + co-cultures) and n = 2 (CD19 − co-cultures) per construct. (F) Flow cytometry analysis of virus-specific T cells (VSTs) electroporated with mRNA constructs encoding for CD19-CAR using various UTRs. Note that cells are grown without target cells to demonstrate tonic signaling. (G) Pie charts of PD-1/TIM-3 population distribution of (F). Data are representative of two independent experiments.

Article Snippet: Cells were harvested 24–48 h post-electroporation, washed in FACS buffer (PBS with 2% FBS), and stained with the following fluorochrome-conjugated antibodies: CD19 CAR Detection Reagent, Biotin (Miltenyi Biotec, #130-129-550), followed by secondary staining with Anti-Biotin-APC (Miltenyi Biotec, REAfinity #130-113-854); TIM-3 APC-Cy7 (BioLegend, #345025); 4-1BB PE-Cy7 (BioLegend, #309820); Viability Dye eFluor 506/AmCyan (Thermo Fisher Scientific, #65-0866-14).

Techniques: Expressing, Flow Cytometry, Derivative Assay, Construct, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Virus

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: JCI Insight

Article Title: A stromal platform for robust expansion of functional IL-10–producing B cells for immune regulation

doi: 10.1172/jci.insight.197393

Figure Lengend Snippet: ( A and B ) Temporal changes in activation markers of B cells cocultured with MS5-3F. ( A ) Representative flow cytometry plots. ( B ) Frequencies and absolute cell numbers of indicated subsets among CD19 + cells. ( C ) Representative histograms and summarized MFI of surface markers and transcriptional factors expressed on CD27 hi and CD27 lo B cells on days 0 and 12 of MS5-3F coculture. See also . ( D ) Morphological analysis of primary B cells (day 0) and MS5-3F–induced B cell subsets, stained with May-Grünwald-Giemsa. CD27 hi and CD27 lo B cells were sorted on day 12 of coculture. Original magnification, ×400. Scale bars: 20 μm. ( E ) Representative flow cytometry plots of IgM, IgD, and IgG on B cells on day 12 of MS5-3F coculture. ( F ) Quantification of IgM and IgG antibody production by cocultured B cells. Culture supernatants were harvested every 4 days and analyzed by ELISA. Data from 3 independent experiments were combined ( B and C : n = 3 or 9; F : n = 9). Data are presented as mean ± SEM. P values are from 1-way ANOVA with Tukey’s post hoc test ( C ). ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. NS, not significant.

Article Snippet: Primary human B cells were isolated from PBMCs by positive selection using CD19 MicroBeads (Miltenyi Biotec).

Techniques: Activation Assay, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay

Journal: JCI Insight

Article Title: A stromal platform for robust expansion of functional IL-10–producing B cells for immune regulation

doi: 10.1172/jci.insight.197393

Figure Lengend Snippet: ( A ) Representative flow cytometry plots of surface markers expressed on B cells from patients with SLE and healthy controls (HCs), and summarized frequencies of memory (CD38 – CD27 + ), naive mature (CD38 – CD27 – ), and naive immature (CD38 lo CD27 – ) B cells among CD19 + cells. See also . ( B ) Summarized frequencies of IL-10 + B cells from patients with SLE and HCs stimulated with CD40L for 2 days (pre-co-culture). B cell stimulation was performed using CD40L-expressing MS5 cells. For the construction of the MS5 cells, see . ( C and D ) Induction and expansion of IL-10 + B cells from patients with SLE and HCs. ( C ) Fold expansion of total B cells from patients with SLE and HCs cocultured with MS5-3F for 12 days. ( D ) Representative flow cytometry plots and summarized frequencies of IL-10 + B cells from SLE and HC B cells, stimulated with CD40L for 2 days (pre-co-culture) or cocultured with MS5-3F for 12 days (post-co-culture). ( E ) ELISA of IL-10 secreted by SLE and HC B cells cocultured with MS5-3F for 12 days. ( F ) ELISA of IgM and IgG secreted by SLE and HC B cells cocultured with MS5-3F for 12 days. Data from 2 independent experiments were combined (SLE: n = 4; HC: n = 8). Data are presented as mean ± SEM. P values are from 2-tailed, unpaired Welch’s t -test ( A – E ) and 1-way ANOVA with Tukey’s post hoc test ( F ). ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. NS, not significant.

Article Snippet: Primary human B cells were isolated from PBMCs by positive selection using CD19 MicroBeads (Miltenyi Biotec).

Techniques: Flow Cytometry, Co-Culture Assay, Cell Stimulation, Expressing, Enzyme-linked Immunosorbent Assay