cd4 t cell kit  (Miltenyi Biotec)

Journal: FEBS Open Bio

Article Title: In vitro generation of regulatory T cells: A challenging tool for studying immune aging in humans

doi: 10.1002/2211-5463.70349

Figure Lengend Snippet: (A) Representative dot plots of isolated peripheral blood mononuclear cells (PBMCs) before (upper row) and after (lower row) total CD4 + cells magnetic‐activated cell sorting (MACS) from a young donor. Cells are gated on the living population and checked for CD4 vs. CD8 (left panels), CD45RA vs. CD28 expression (middle panels), and for CD127 vs. CD25 (right panels) expression. Frequencies of CD4 + Foxp3 + (B) and CD25 + of CD4 + Foxp3 + (C) cells are shown after 4 days of in vitro induction of Treg cells in the presence of 10% fetal calf serum (FCS) containing medium RPMI or in serum‐free medium XVivo‐15 (MEAN; n = 2, young donors).

Article Snippet: For the purification of total CD4 + T cells, we used the Miltenyi Biotec CD4 T‐cell Kit (130‐096‐533) according to the manufacturer's protocol.

Techniques: Isolation, FACS, Expressing, In Vitro

Journal: FEBS Open Bio

Article Title: In vitro generation of regulatory T cells: A challenging tool for studying immune aging in humans

doi: 10.1002/2211-5463.70349

Figure Lengend Snippet: Frequency of Foxp3 + CD4 + cells (A) and CD25 expression in Foxp3 + cells (B) after 6 days of induction in TCR‐stimulated cells in the presence of IL‐2 (iTreg IL‐2 ) only or IL‐2 + TGF‐β (iTreg TGFβ ) in young and older individuals. (Mean ± SEM; n ≥ 3). Statistical significance P < 0.05*, and P < 0.01** (two‐way ANOVA with Sidak's post hoc test).

Article Snippet: For the purification of total CD4 + T cells, we used the Miltenyi Biotec CD4 T‐cell Kit (130‐096‐533) according to the manufacturer's protocol.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: A fully human afucosylated anti-GARP antibody PBA-0111 drives anti-tumor immunity through combined ADCC induction and TGF-β1 pathway blockade

doi: 10.3389/fimmu.2026.1921265

Figure Lengend Snippet: PBA-0111 demonstrates potent ADCC against GARP and GARP/TGF-β1 positive tumor cells and Tregs, accompanied by NK cell activation. (A) Raji-GARP target cells were treated with serial dilutions (0.00001-100 nM) of PBA-0111, DS-1055a-like antibody, or afucosylated isotype control and subjected to NK cell-mediated lysis for 4 h at 37 °C at an Effector: Target ratio of 10:1. Cell killing was analyzed by flow cytometry using live/dead stain. Data are shown as mean ± SD from a representative pool of three independent donors tested in duplicate. (B) ADCC was performed against Raji-GARP/TGF-β1 target cells (n=6, from 3 independent donors) using the same conditions as in (A) . (C) ADCC against the L-428 lymphoma cell line (n=6, from 3 independent donors). (D) ADCC against in vitro differentiated Tregs. CD4+CD25high T cells isolated from healthy donor PBMCs were stimulated with anti-CD3/CD28 beads to generate regulatory T cells, then subjected to autologous NK-mediated cellular lysis overnight at 37 °C in the presence of indicated anti-GARP antibodies. Data represent pooled results from three independent donors tested in duplicate, mean ± SD. (E) PBMCs (n=3 donors, in 2 replicates) were co-incubated with Raji-GARP/TGF-β1 cells and either PBA-0111, DS-1055a-like antibody, or afucosylated isotype control (IgG.aF) at 5 nM for 4 hours. NK cells were then analyzed by flow cytometry for CD107a expression in the presence of Golgi stop. (F) The same NK cells were analyzed for IFN-γ and (G) TNF-α production. Data are expressed as fold change relative to the afucosylated isotype control. Statistical significance versus IgG1.aF was assessed by Kruskal-Wallis with Dunn´s multiple comparison, *p< 0.05, **p< 0.01, ***p< 0.001.

Article Snippet: Regulatory T cells were isolated from PBMCs from the same donor as NK cells by immunomagnetic two-step procedure with human CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, 130-091-301) according to the manufacturer’s protocol.

Techniques: Activation Assay, Control, Lysis, Flow Cytometry, Staining, In Vitro, Isolation, Incubation, Expressing, Comparison

Journal: Frontiers in Immunology

Article Title: A fully human afucosylated anti-GARP antibody PBA-0111 drives anti-tumor immunity through combined ADCC induction and TGF-β1 pathway blockade

doi: 10.3389/fimmu.2026.1921265

Figure Lengend Snippet: PBA-0111 activates T cells leading to anti-tumor efficacy. (A–D) CD34 + HSC humanized NSG-hIL15 mice (n=6-7/group) were inoculated subcutaneously with Raji-GARP/TGF-β1 and treated with vehicle or six doses of PBA-0111 or DS-1055a-like antibody at 10 mg/kg twice weekly, initiated when the tumor volumes reached 80-150 mm 3 . At study termination, tumors, spleens and terminal blood were collected and processed for FACS analysis. (A) Proportion of CD8 + cells within the tumor (B) Intratumoral CD8 + /Treg ratio. (C) Activation status of intratumoral CD8 + T cells. (D) NK cell activation in peripheral blood. (E–I) CD34 + HSC humanized NCG-hIL15 mice (n=8/group) were inoculated subcutaneously with HT-29 colon carcinoma cells and treated as above. At study termination, tumors and terminal blood were processed for FACS analysis (n=4-8/group). (E) Proportion of intratumoral CD8 + T cells. (F) Intratumoral CD8+/Treg ratio. (G) CD8 + T cell infiltration confirmed by immunohistochemistry (IHC). (H) Granzyme B expression detected by IHC. (I) Representative IHC images showing CD8 + (green) and Granzyme B + (magenta) cells in immune-positive clusters at the tumor periphery (rim) of HT-29 tumors. Scale bar = 100 µm. Statistical significance was assessed by Kruskal-Wallis test with Dunn´s multiple comparisons. **p < 0.01.

Article Snippet: Regulatory T cells were isolated from PBMCs from the same donor as NK cells by immunomagnetic two-step procedure with human CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, 130-091-301) according to the manufacturer’s protocol.

Techniques: Analysis, Activation Assay, Immunohistochemistry, Expressing

Journal: Frontiers in Immunology

Article Title: A fully human afucosylated anti-GARP antibody PBA-0111 drives anti-tumor immunity through combined ADCC induction and TGF-β1 pathway blockade

doi: 10.3389/fimmu.2026.1921265

Figure Lengend Snippet: PBA-0111 shows signs of T cell activation in soft tissue sarcoma patients following intratumoral administration. (A) CIVO workflow. PBA-0111 was co-formulated with fluorescent microspheres and loaded into the CIVO injector, which delivers eight individual columns of drug directly into the patient tumor (left). STS patients receiving the multiplexed microinjection were evaluated immediately following the procedure by visualization of individual injection sites using a blue light and yellow filter that illuminates the CIVO GLO fluorescent microspheres with an ultraviolet flashlight (center). Following a pre-scheduled surgical resection 2-96 hours later, the tumors were sectioned transverse to the drug columns, yielding 2-4 sections of FFPE tissue for downstream IHC and ISH staining and image analysis. (B) GARP expression assessed by ISH. Representative injection site images from vehicle and PBA-0111-treated sites in Patient 5, with summary data showing the median percentage of GARP + cells across all evaluable injection sites in all patients (n=6). (C) Regulatory T cell infiltration assessed by IHC. Representative injection site images for vehicle and PBA-0111-treated sites in Patient 5, with summary data showing the median percentage of CD4+ T cells that are FoxP3 + across all evaluable injection sites in all patients. (D) . CD8 + cytotoxic T cell activity assessed by IHC. Representative injection site images for vehicle and PBA-0111-treated sites in Patient 5, with summary data showing the median percentage of CD8 + T cells that are Granzyme B + across all evaluable injection sites in all patients.

Article Snippet: Regulatory T cells were isolated from PBMCs from the same donor as NK cells by immunomagnetic two-step procedure with human CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, 130-091-301) according to the manufacturer’s protocol.

Techniques: Activation Assay, Tissue, Microinjection, Injection, Staining, Analysis, Expressing, Activity Assay