Journal: Journal of Nanobiotechnology

Article Title: MiR-1290 in natural killer cell derived extracellular vesicles: a pathogenic mediator of lupus nephritis and therapeutic target for th17 regulation

doi: 10.1186/s12951-026-04212-9

Figure Lengend Snippet: Characterization of NK-EVs and their effect on Th17 cell differentiation in vitro. (a) Schematic of the experimental procedure for treating primary T cells with NK-EVs. (b) Purity analysis of sorted CD4 + T cells by flow cytometry. (c) Purity analysis of sorted NK cells, showing the proportion of CD3 − CD56 + NK cells exceeded 94%. (d) Size distribution of NK-EVs as determined by NTA. (e) Western blot analysis of exosomal markers and specific cargos in NK-EVs, with 293T-derived EVs as a control. (f) TEM analysis of NK-EVs showing cup-shaped bilayer membrane structures (indicated by black arrows, scale bar: 200 nm). (g) Immunofluorescence staining showing the uptake of NK-EVs by T cells (indicated by black arrows, scale bar: 10 μm) and (h) fluorescence intensity analysis of the local region indicated by white arrows. (i) Gating strategy and representative flow cytometry plots for CD4 + IL-17 A + T cells under Th17-polarizing conditions after EVs treatment. (j) LN-EVs significantly reduced the proportion of CD4 + IL-17 A + cells ( N = 3). (k) Schematic of the procedures for generating Protein-free NK-EVs via protease treatment and RNA-free NK-EVs via RNase treatment. (l) Effects of LN-derived NK-EVs, Protein-free NK-EVs, and RNA-free NK-EVs on the proportion of Th17 cell differentiation. (m) Effects of LN-derived NK-EVs, Protein-free NK-EVs, and RNA-free NK-EVs on the IL17A mRNA level in T cells. (n) Effects of LN-derived NK-EVs, Protein-free NK-EVs, and RNA-free NK-EVs on the levels of inflammatory cytokines IFN-γ, TNF-α, IL-17 A, and IL-10 in T cell culture supernatants. Data are presented as mean ± SD ( N = 3). * P < 0.05, ** P < 0.01 (One-way ANOVA)

Article Snippet: The resulting cell fraction was then subjected to positive selection using human CD56 MicroBeats (Miltenyi Biotec, #130-050-401), yielding a highly pure CD3−CD56+ NK cell population (identified by flow cytometry) [ ].

Techniques: Cell Differentiation, In Vitro, Flow Cytometry, Western Blot, Derivative Assay, Control, Membrane, Immunofluorescence, Staining, Fluorescence, Cell Culture

Journal: Cell Transplantation

Article Title: A novel GMP-manufactured medicinal product candidate composed of NK and γδ T cells as adjunct immunotherapy for hematopoietic stem cell transplantation

doi: 10.1177/09636897251374248

Figure Lengend Snippet: Flow cytometric analysis of cell populations during the process. Panel A. Number of cells and proportion of populations obtained at the different steps of the process starting from buffy coat (BC) or leukapheresis (LA). Panels B–D. One representative experiment using LA (#3) as staring material is shown. Panel B. CD14 + monocytes (a), CD19 + B cells (b) CD3 + T cells (c), CD3 − CD56 + NK cells (d), γδ and αβ T lymphocytes (e) as well as Vδ1 and Vδ2 subsets (f), present after Ficoll-Paque gradient in gated lympho-mononuclear cells. Panels C and D. CD14 + monocytes (a), CD19 + B cells (b) CD3 − CD56 + NK cells (c), γδ and αβ T lymphocytes (d) as well as Vδ1 and Vδ2 subsets (e), gated mononuclear cells at the end of expansion (day 14) (Panel C) and harvested after αβ T-cell depletion (Panel D). Panel E shows γδ T cell differentiation, by APC-CD27 and PEVio770-CD45RA staining.

Article Snippet: Immunophenotype was assessed on at least 50,000 cells by flow cytometry using fluorochrome-conjugated monoclonal antibodies (mAb): FITC-CD3, PerCP-CD14, Viogreen-CD19, PEVio770 anti-TCRαβ, APC anti-TCRγδ, Viogreen-TCRVδ1, Vioblue-TCRVδ2, and APC-CD56 (all from Miltenyi).

Techniques: Cell Differentiation, Staining

Journal: Cell Transplantation

Article Title: A novel GMP-manufactured medicinal product candidate composed of NK and γδ T cells as adjunct immunotherapy for hematopoietic stem cell transplantation

doi: 10.1177/09636897251374248

Figure Lengend Snippet: Cytotoxic activity of the GADEKILL against target tumor cells. CD107a surface expression on γδ T lymphocytes (Panel a), Vδ2 cells (Panel b), and NK cells (Panel c) in the absence (ctr) or presence of target cells (K562, SK-N-AS and SK-N-SH) have been analyzed on the four batches. γδ T cells and Vδ2 cytotoxic subsets were analyzed in gated CD45 + CD3 + cells, by staining with APC-TCRγδ, Vioblue anti-Vδ2. NK were analyzed on gated CD45 + CD3 − cells, by staining with APC-CD56 mAb. CD107a degranulation assay was performed using a 1:1 E:T ratio. Horizontal bars indicated medians. Asterisks indicated statistically significant differences. One representative experiment from batch #2 is shown in panels d, e and f. Degranulation of effector cells in the absence of the targets (Panel d) and against the myeloid leukemia K562 cells (Panel e) and the neuroblastoma SK-N-AS cell line (Panel f) is reported.

Article Snippet: Immunophenotype was assessed on at least 50,000 cells by flow cytometry using fluorochrome-conjugated monoclonal antibodies (mAb): FITC-CD3, PerCP-CD14, Viogreen-CD19, PEVio770 anti-TCRαβ, APC anti-TCRγδ, Viogreen-TCRVδ1, Vioblue-TCRVδ2, and APC-CD56 (all from Miltenyi).

Techniques: Activity Assay, Expressing, Staining, Degranulation Assay

Journal: Frontiers in Immunology

Article Title: Human serum influences functional plasticity and transcriptomic landscape of γδ T cells in vitro

doi: 10.3389/fimmu.2026.1722590

Figure Lengend Snippet: Impact of serum on γδ T cell expansion and phenotype. (A) Fold expansion comparison with and without 5% human AB serum in the culture medium, analyzed using the Wilcoxon matched pairs signed rank test, n=14. (B) Cellular composition assessed by flow cytometry throughout the expansion, illustrated for a representative donor. (C) γδ T cell phenotype determined by flow cytometry based on CD27 and CD45RA expression. Exemplary plots on day 13 and proportions throughout the culture for a representative donor. (D) Expression of activation markers on γδ T cells analyzed by flow cytometry. Šídák’s multiple comparisons test, n=15 for CD69, CD56, and HLA-DR; n=6 for NKG2D. (E) Expression of inhibitory and exhaustion markers on γδ T cells, analyzed by flow cytometry. Šídák’s multiple comparisons test, n=15. In the figures, the significance levels denoted by stars are as follows: *p < 0.05, ***p < 0.001, ****p < 0.0001, ns = non-significant.

Article Snippet: The following antibodies were used for cell surface staining and intracellular staining: anti-human CD107a REA803, anti-human CD14 REA599, anti-human CD19 REA675, anti-human CD27 REA499, anti-human CD3 REA613, anti-human CD45RA REA562, anti-human CD56 REA196, anti-human CD69 REA824, anti-human Granzyme B REA226, anti-human HLA/DR REA805, anti-human IFNγ 45-15, anti-human KIR2D REA1042, anti-human NKG2D REA797, anti-human PD-1 REA1165, anti-human Perforin REA1061, anti-human REA Control (I) REA293, anti-human REA Control (S) REA293, anti-human TCR Vδ1 REA173, anti-human TCR Vδ2 REA771, anti-human TCR γδ REA591, anti-human TIGIT REA1004, and anti-human TIM3 F38-2E2 (all from Miltenyi Biotec).

Techniques: Comparison, Flow Cytometry, Expressing, Activation Assay