Journal: Frontiers in Immunology

Article Title: Trifunctional antibody fusion proteins for targeted co-delivery of 4-1BBL with IL-15 or IL-21 to enhance antitumor immune responses

doi: 10.3389/fimmu.2026.1903525

Figure Lengend Snippet: Trifunctional fusion proteins enhance cytotoxic potential differently. Fusion proteins were incubated (A) untargeted or (B) targeted to B16-FAP cells (preincubated with the indicated concentration and then washed to remove unbound fusion protein) with PBMCs in presence of suboptimal concentration of CytoStim™. After 4 days, cells were fixed, permeabilized and intracellularly stained with anti-CD3, anti-CD8 and anti-granzyme B antibodies, followed by flow cytometry analysis. Graphics show mean ± SD, n=3, independent experiments, using PBMCs from the same donor and fusion proteins from batches #2, with blockshift correction, One-way ANOVA, Tukey’s post-test **p<0.01; ***p<0.001; ****p<0.0001.

Article Snippet: Antibodies and other reagents were purchased from BioLegend (anti-CD3 PE, 317308; anti-CD8 Pacific Blue, 344718; anti-CD56 APC, 362504; anti-CD69 FITC, 310904; anti-CD56 PacificBlue, 362519; FoxP3 Fix/Perm buffer,421401; FoxP3 Perm buffer, 421402), Miltenyi Biotech (anti-His-PE, 130-120-718; anti-CD8 PEVio770.

Techniques: Incubation, Concentration Assay, Staining, Flow Cytometry, Analysis

Journal: Frontiers in Immunology

Article Title: UPF1-dependent glycolysis driven by LINC00887 facilitates macrophage M2 polarization in clear cell renal cell carcinoma

doi: 10.3389/fimmu.2026.1833383

Figure Lengend Snippet: Overexpression of UPF1 in ccRCC inhibits M2 macrophage polarization and enhances the tumor suppressor function of CD8 + T cells, Caki-1 cell groups of sh-NC+PBS, sh-UPF1+PBS, or sh-UPF1 + 2-DG, and the conditioned medium was used to culture and polarize M0 macrophages. The polarized macrophages were cultured with activated CD8 + T cells. (A) CFSE assessed CD8 + T cell proliferative capacity. (B) Flow cytometry measured apoptosis rate of CD8 + T cells in different co-culture systems. (C) CD8 + T cells from culture systems were co-cultured with normal Caki-1 cells, with LDH assays determining cytotoxic effect of CD8 + T cells. *, p < 0.05. cRCC, Clear cell renal cell carcinoma; UPF1, Up-frameshift protein 1; 2-DG, 2-Deoxy-D-glucose; CFSE, Carboxyfluorescein succinimidyl ester.

Article Snippet: Antibodies included: FITC Mouse monoclonal to CD68 (ab134351, abcam, UK), Pacific Blue anti-human CD86 Antibody (305417, yajimall, China), CD206 (MMR) Monoclonal Antibody (19.2)-PE (12-2069-42, ThermoFisher, USA), PerCP/Cyanine5.5 Anti-Mouse F4/80 Antibody (E-AB-F0995J, Elabscience, China), PE Anti-CD206 mouse antibody (E-AB-F1135D, Elabscience, China), APC Anti-mouse CD3 antibody (E-AB-F1013E, Elabscience, China), PE Anti-mouse CD8 alpha antibody (E-AB-F1104D, Elabscience, China).

Techniques: Over Expression, Cell Culture, Flow Cytometry, Co-culture Assay

Journal: Frontiers in Immunology

Article Title: UPF1-dependent glycolysis driven by LINC00887 facilitates macrophage M2 polarization in clear cell renal cell carcinoma

doi: 10.3389/fimmu.2026.1833383

Figure Lengend Snippet: LINC00887/UPF1-mediated aerobic glycolysis leads to lactic acid accumulation and facilitates immune evasion in ccRCC in vivo , A syngeneic tumor model was developed by injecting BALB/c mice with Renca cells expressing sh-NC or sh-LINC00887, followed by treatment with sh-NC or sh-UPF1 lentivirus. A-C: Assessment of tumor size (A) , volume (B) , and weight (C) . (D, E) qRT-PCR analyses for LINC00887 (D) and UPF1 (E) mRNA levels. (F) IHC analysis for UPF1, LDH-A, KI67 protein levels. (G) Kit assay for lactate levels in tumors. (H) Proportion of M2 macrophages (F4/80+CD206+) by flow cytometry. (I) Immunofluorescence assay for CD206+ macrophages. (J) Proportion of CD8 + T cells (CD3 + CD8 + ) in tumor tissues. ns, no significant difference. * p < 0.05. cRCC, Clear cell renal cell carcinoma; UPF1, Up-frameshift protein 1; LDH-A, Lactate dehydrogenase A; CD206, Mannose receptor C-type 1; CD3, Cluster of differentiation 3; CD8, Cluster of differentiation 8.

Article Snippet: Antibodies included: FITC Mouse monoclonal to CD68 (ab134351, abcam, UK), Pacific Blue anti-human CD86 Antibody (305417, yajimall, China), CD206 (MMR) Monoclonal Antibody (19.2)-PE (12-2069-42, ThermoFisher, USA), PerCP/Cyanine5.5 Anti-Mouse F4/80 Antibody (E-AB-F0995J, Elabscience, China), PE Anti-CD206 mouse antibody (E-AB-F1135D, Elabscience, China), APC Anti-mouse CD3 antibody (E-AB-F1013E, Elabscience, China), PE Anti-mouse CD8 alpha antibody (E-AB-F1104D, Elabscience, China).

Techniques: In Vivo, Expressing, Quantitative RT-PCR, Analysis, Flow Cytometry, Immunofluorescence

Journal: Nature Medicine

Article Title: Multi-antigen-targeting T cells in pediatric central nervous system tumors: a phase 1 trial

doi: 10.1038/s41591-026-04449-9

Figure Lengend Snippet: Flow immunophenotyping release testing data are presented in A-G and IFNγ ELISpot data presented in H-K (Arm A: n = 12; Arms B/C: n = 27). Each data point represents a separate TAA-T cell product infused into patients (biological replicates). Lower whisker is defined as the minima, upper whisker is defined as the maxima, center is defined as median, lower bound of box is defined as Q1 (25th percentile), and upper bound of box is defined as Q3 (75th percentile). a ) % Total T cells (CD3 + ). Arm A: Minima=51, Maxima=99, Median=97, 25th Percentile=95, 75th Percentile=99; Arms B/C: Minima=31, Maxima=100, Median=97, 25th Percentile=94, 75th Percentile=98. b ) % CD4 + T cells (CD3 + CD4+ of CD45 + ). Arm A: Minima=0.50, Maxima=88, Median=16, 25th Percentile=2.6, 75th Percentile=38; Arms B/C: Minima=0.40, Maxima=55, Median=9.9, 25th Percentile=6.1, 75th Percentile=17. c ) %CD8 + T cells (% CD3 + CD8+ of CD45 + ). Arm A: Minima=4.3, Maxima=88, Median=37, 25th Percentile=22, 75th Percentile=47; Arms B/C: Minima=19, Maxima=91, Median=51, 25th Percentile=36, 75th Percentile=71. d ) % αβ T cells (% TCRαβ+ of CD3 + ). Arm A: Minima=8.2, Maxima=94, Median=69, 25th Percentile=28, 75th Percentile=86; Arms B/C: Minima=32, Maxima=98, Median=71, 25th Percentile=48, 75th Percentile=84. e ) % γδ T cells (% TCRγδ+ of CD3 + ). Arm A: Minima=5.1, Maxima=79, Median=28, 25th Percentile=8.4, 75th Percentile=67; Arms B/C: Minima=1.1, Maxima=83, Median=25, 25th Percentile=10, 75th Percentile=53. f ) % CD3 + CD16 + CD56+ of CD45 + . Arm A: Minima=2.8, Maxima=25, Median=11, 25th Percentile=7.2, 75th Percentile=17; Arms B/C: Minima=3.4, Maxima=64, Median=16, 25th Percentile=8.1, 75th Percentile=28. g ) % NK cells (CD16/CD56 + CD3- of CD45 + ). Arm A: Minima=0.40, Maxima=48, Median=1.0, 25th Percentile=0.48, 75th Percentile=4.6; Arms B/C: Minima=0.10, Maxima=62, Median=1.6, 25th Percentile=0.60, 75th Percentile=6.1. h - i ) IFNγ ELISpot with background correction for Arm A ( h ) and Arms B/C ( i ). Negative values after background normalization have been normalized to zero for visual representation.

Article Snippet: Antibodies for the T/NK panel included CD8 FITC (Miltenyi Biotec, cat. no. 130-110-677), CD16 PE (Miltenyi Biotec, cat. no. 130-113-393), CD56 PE (Miltenyi Biotec, cat. no. 130-113-312), CD3 Per CP Vio-770 (Miltenyi Biotec, cat. no. 130-113-141), CD4 PE-Vio-770 (Miltenyi Biotec, cat. no. 130-113-227) and CD45 APC (Miltenyi Biotec, cat. no. 130-110-633).

Techniques: Enzyme-linked Immunospot, Whisker Assay

Journal: International Journal of Molecular Sciences

Article Title: Proinflammatory Cytokine Preconditioning Enhances the Therapeutic Potency of Different Types of MSCs in Inflammation

doi: 10.3390/ijms27094090

Figure Lengend Snippet: The effects of naïve and Cytomix-stimulated MSCs on T cell proliferation. The gating strategy is shown in . ( A – C ) BM-MSCs, iMSCs WT and iMSCs B2M KO inhibition on the expansion of CD3+, CD3+CD4+ and CD3+CD8+ T cells via CM. Cytomix enhanced the three types of MSCs’ inhibition on the EI of T cells. ( D – F ) All types of MSCs significantly inhibited the division of CD3+, CD3+CD4+ and CD3+CD8+ T cells when co-cultured with PBMCs, in a dose-dependent manner. ( G – I ) All of the MSCs, naïve or Cytomix stimulated, significantly reduced the EI of CD3+, CD3+CD4+ or CD3+CD8+ T cells, at the lowest ratio of MSCs, with iMSCs WT both naïve and Cytomix-stimulated presenting the strongest inhibition. ( J ) Co-culturing all types of MSCs, naïve or Cytomix-stimulated, increased the production of G-CSF in the media, with a dose-dependent effect. ( K ) All types of MSCs significantly decreased the level of IFN-γ at the ratios of 1:20 and 1:10, while the three types of naïve MSCs significantly decreased it at 1:50. ( L ) Naïve and Cytomix-stimulated iMSC WTs significantly increased the level of TNF-α at the ratios of 1:50, 1:20 and 1:10, while Cytomix-stimulated iMSC B2M KO significantly increased it at the ratio of 1:50 and 1:20. ( M ) Naïve BM-MSCs significantly increased the level of IL-10 production at the ratios of 1:20 and 1:10. Representative results of four independent experiments are shown. Results are shown as mean ± SD ( n = 3 in CM; n = 4 in co-culture, each group), and analysed by two-way ANOVA with Šídák’s multiple comparisons test to compare naïve vs. Cytomix in each type of MSCs (no significance in all of the settings), or Tukey’s multiple comparisons test to compare different ratios of MSCs to PBMCs in each MSC group, or Dunnett’s multiple comparisons test to compare different MSC groups vs. vehicle in the setting of the same ratio. */**/***/**** represent comparisons of each type of MSC vs. vehicle; #/## represent comparisons of different ratios of each cell type with different levels of p < 0.05/0.01. EI: expansion index; DP: division percentage.

Article Snippet: The PBMCs were stained with anti-human CD3-APC-Vio770, CD4-APC and CD8-PE-Vio770 antibodies (Cat# 130-113-136, 130-113-222, 130-110-680; Miltenyi Biotec) for 30 min on ice in the dark.

Techniques: Inhibition, Cell Culture, Co-Culture Assay