Journal: Science Advances

Article Title: Conserved noncoding sequence-9 regulates NFATc1-mediated IL-10 expression in B cells to control inflammatory responses

doi: 10.1126/sciadv.aec7779

Figure Lengend Snippet: ( A ) Analysis of ChIP-seq data of p300 (black), histone (blue), NFATc1 (green), and in situ Hi-C data at the IL10 locus using public datasets ( GSE32465 , GSE29611 , and GSE63525 ) of GM12878 human immortalized B cells. ( B to E ) Raji B cells were electroporated with vectors expressing Cas9 and sgRNAs targeting NFAT-binding motifs in IL10 CNS-12 or mock vectors. (B) Schematic diagram of the NFAT-binding motifs in IL10 CNS-12, identified with rVISTA (Transfac matrices, similarity score of 0.85). Red arrows indicate sgRNA targeting sites. (C) qRT-PCR analysis of IL10 mRNA in electroporated human Raji B cells, expressed as fold change relative to unstimulated cells. [(D) and (E)] Representative flow cytometry plots (D) and frequency (E) of IL-10 + cells in Raji B cells ( n = 3 for mock, sgRNA 1, and sgRNA 2). A FMO control for IL-10 is shown in (D). ( F ) IL-10 concentrations in culture supernatants of Raji B cells measured at 4 and 24 hours ( n = 3 for mock, sgRNA 1, and sgRNA 2). Data are pooled from three independent experiments [(C), (E), and (F)]. Data are presented as means ± SEM. Statistical analysis was performed using a two-tailed unpaired Student’s t test [(C), (E), and (F)]: * P < 0.05, ** P < 0.01, and **** P < 0.0001.

Article Snippet: A20 and Raji B cell lines were obtained from the American Type Culture Collection (MD, USA).

Techniques: ChIP-sequencing, In Situ, Hi-C, Expressing, Binding Assay, Quantitative RT-PCR, Flow Cytometry, Control, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Erythrocyte membrane–liposome coating sustains circulation stability and targeted tumor therapy of CAR-T cells

doi: 10.3389/fimmu.2026.1799107

Figure Lengend Snippet: Impact of Rlip modification on CD19 CAR-T cell binding efficiency, CAR expression, and functional phenotype. (A) . Schematic of the anti-CD19 CAR construct. (B) . Flow-cytometry histograms showing CD19 CAR expression in untransduced human T cells versus anti-CD19 CAR-T cells. (C). Flowcytometry histograms of RhB positivity in CAR-T and Rlip-CAR-T cells (reflecting Rlip binding efficiency). (D) . Flow-cytometry dot plots showing CD19 CAR expression levels in CAR-T and Rlip-CAR-T cells. (E) . Quantitative analysis of CD19 CAR expression ratios between CAR-T and Rlip-CART cells. (F) . Cytotoxic activity of CAR-T and Rlip-CAR-T cells against Nalm-6 and Raji cells at the indicated E:T ratios. (G) . Flow-cytometry dot plots showing memory-phenotype distribution of CAR-T and Rlip-CAR-T cells stained for CD62L and CD45RA. (H) . Quantitative analysis of T-cell memory subsets (TSCM, TCM, TEM, TTE) in CAR-T and Rlip-CAR-T cells. (I) . Quantitative analysis of activation-marker (CD69, CD25) expression in CAR-T and Rlip-CAR-T cells. (J) . Quantitative analysis of exhaustion-marker (PD-1, LAG-3) expression in CAR-T and Rlip-CAR-T cells. All data are obtained from at least three donors and presented as mean ± SD; ns, not significant. Cytotoxic activity (F) was analyzed by two-way ANOVA; all other quantitative comparisons (E, H–J) used two-tailed unpaired t-tests.

Article Snippet: Human breast cancer cell line HCC1806, ovarian cancer cell line OVCAR3, B-cell lymphoma cell line Raji, human monocytic leukemia cell line THP-1, and B-cell precursor leukemia cell line NALM-6 were purchased from the American Type Culture Collection (ATCC).

Techniques: Modification, Binding Assay, Expressing, Functional Assay, Construct, Flow Cytometry, Activity Assay, Staining, Activation Assay, Marker, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Glycoengineering CAR-T cells to overcome galectin-3-mediated immunosuppression

doi: 10.3389/fimmu.2026.1766555

Figure Lengend Snippet: ST6 OE CAR-T cells exhibited effective tumoricidal activity in the presence of Gal-3 and evaded Gal-3-induced expression of IL-5, which compromised anti-tumor efficacy. Day 10-expanded T cells, CAR-T cells, and ST6 OE CAR-T cells were incubated with Gal-3 low Raji-luc + cells or Gal-3 high SUDHL-6-luc + cells at effector:target (E:T) ratios of 20:1. Raji cell cytotoxicity was evaluated (a) without rhGal-3 or (b) with rhGal-3, and (c) Gal-3 high SUDHL-6 cell cytotoxicity was examined without exogenous rhGal-3. Data are presented from n = 4 independent donors and analyzed using a two-way repeated-measures ANOVA with Sidak’s multiple-comparisons test, with ** p < 0.01, * p < 0.05. (d) Human cytokine proteome profiling of supernatants from Day 10-expanded T cells, CAR-T cells, and ST6 OE CAR-T cells incubated with rhGal-3 for 16h revealed a marked upregulation of IL-5 (red hatched box) in CAR-T cells that was absent in ST6 OE CAR-T cells. (e) Arrays were repeated 4 times and graphed as Relative Optical Density (Mean ± SEM). Intracellular IL-5 expression was assessed by flow cytometry in control Day 10-expanded T cells, CAR-T cells and ST6 OE CAR-T cells after rhGal-3 incubation for 16h and mean ± SEM fold change from n = 4 independent donors was graphed (f) (* p < 0.05). Statistical significance was determined using a Mann–Whitney U test. CAR-T cells and ST6 OE CAR-T cells were incubated with (g) Gal-3 low Raji-luc + cells or (h) Gal-3 high SUDHL6-luc + at E:T ratios of 20:1 (** p <0.01, *** p <0.001). Statistical significance was determined using two-way repeated-measures ANOVA with Sidak’s multiple-comparisons test.

Article Snippet: Human B-lymphoma Raji cell line expressing luciferase (Raji-Luc + ) purchased from the ATCC (Manassas, VA).

Techniques: Activity Assay, Expressing, Incubation, Flow Cytometry, Control, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Glycoengineering CAR-T cells to overcome galectin-3-mediated immunosuppression

doi: 10.3389/fimmu.2026.1766555

Figure Lengend Snippet: ST6 OE CAR-T cells exhibited potent in vivo anti-tumor activity and persisted longer in vivo than conventional CAR-T cells. Using a DLBCL xenograft model (a) , NSG mice were inoculated with Raji-luc + cells and, after 7 days, treated with media (control), Day 10-expanded T cells, CAR-T cells, or ST6 OE CAR-T cells. Tumor growth in NSG mice was monitored weekly by in vivo BL imaging system (b) , and BLI measurements reflecting tumor burden from respective groups were graphed as shown (n = 9 mice per group) (** p < 0.01) (c) . Xenografted mouse survival data were graphed as shown (Gehan-Breslow-Wilcoxon test – ** p = 0.0106) (d) , and flow cytometry of CAR + T cells isolated from xenografted mouse spleens expressed as Mean ± SEM were graphed as shown (Mann–Whitney U test- * p <0.05) (e) .

Article Snippet: Human B-lymphoma Raji cell line expressing luciferase (Raji-Luc + ) purchased from the ATCC (Manassas, VA).

Techniques: In Vivo, Activity Assay, Control, Imaging, Flow Cytometry, Isolation, MANN-WHITNEY