cd4 cd8 til microbeads  (Miltenyi Biotec)

Journal: iScience

Article Title: Fluorescence tracking Treg movement identifies anti-CCR8 and radiation as a therapeutic combination

doi: 10.1016/j.isci.2025.114572

Figure Lengend Snippet: Tregs traffic from the tumor to the TdLN at higher proportions and are equally susceptible to radiation as non-Treg CD4 T cells (A) Kaede mice were injected with either MC38, MOC1, or MOC2 cancer cells subcutaneously. Tumors were photoconverted on day 14 via ultraviolet light. TdLNs were harvested on day 3 post-photoconversion. All cell populations were pre-gated on live singlets. (B) Photoconverted CD8 T cells were defined as CD3 + CD4 − CD8 + Red + . Photoconverted Tregs were defined as CD3 + CD8 − CD4 + CD25 + Red + . Photoconverted non-Treg CD4 T cells were defined as CD3 + CD8 − CD4 + CD25 − Red + . (C) Frequencies of photoconverted CD8 T cells, Tregs, and non-Treg CD4 T cells were quantified via flow cytometry. (D) Mice were injected with MC38 cancer cells subcutaneously. Tumors were treated with 12 Gy RT on day 14 and harvested for analysis via flow cytometry on day 1–3 post-RT. Non-Treg CD4 T cells and Tregs frequencies as a percentage of live cells were quantified. (E) MC38-bearing Kaede mouse tumors were photoconverted on day 14 followed by immediate treatment with 12 Gy RT. (F) TdLNs were harvested on day 1–3 and percentages of photoconverted non-Treg CD4 T cells and Tregs were analyzed via flow cytometry. A total of 5–6 mice were analyzed per group. Data are represented as mean ± SD. Statistics were performed using an unpaired Student’s t test between groups. ns = not significant, ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05.

Article Snippet: CD4/CD8 (TIL) MicroBeads, mouse , Miltenyi Biotec , Cat #: 130-116-480.

Techniques: Injection, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: An innovative engineered IL-10 monomer strengthens T cell-mediated anti-tumor responses through anti-PD-1 cis -delivery

doi: 10.1016/j.xcrm.2025.102515

Figure Lengend Snippet: IL-10 exerts anti-tumor efficacy in vivo and promotes erythrocyte phagocytosis in vitro (A) Balb/c-hPD-1 mice were inoculated with 1 × 10 5 CT26 tumor cells. Tumor-bearing mice ( n = 7/group) were intraperitoneally treated with indicated proteins on days 9, 13, 16, and 20. The tumor volume of mice was measured as indicated. Statistical analyses were performed by two-way ANOVA with Dunnett’s multiple comparisons tests (∗ p ≤ 0.05; ∗∗∗∗ p ≤ 0.0001). (B) PBMCs were incubated with WT IL-10-Fc in vitro . Protein binding to CD4 + T, CD8 + T, NK, or CD14 cells was detected by flow cytometry (FCM). Statistical analyses were performed by one-way ANOVA with Dunnett’s multiple comparisons tests (∗∗∗∗ p ≤ 0.0001). Data are representative of three independent experiments performed on different donors. (C) Monocytes were isolated from PBMCs and induced into macrophages using 50 ng/mL GM-CSF. The expression of CD11b, CD16, or CD163 was detected by FCM analysis. Statistical analyses were performed by unpaired Student’s t tests. (∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001). Data are representative of two independent experiments. (D) RBCs were isolated from PBMCs and pre-incubated with IgG antibody for 30 min. The phagocytic activity of macrophages toward RBCs was detected by FCM analysis. Statistical analyses were performed by one-way ANOVA with Dunnett’s multiple comparisons tests. (∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001). Results are reported as the mean ± SEM. Data are representative of three independent experiments. The hPD-1 knock-in humanized mouse is a model in which the mouse PD-1 gene is partially replaced with the human PD-1 gene using gene-editing technology, enabling the expression of human PD-1 protein.

Article Snippet: CD8 (TIL) MicroBeads, mouse , Miltenyi Biotec , Cat#: 130-116-478.

Techniques: In Vivo, In Vitro, Incubation, Protein Binding, Flow Cytometry, Isolation, Expressing, Activity Assay, Knock-In

Journal: Cell Reports Medicine

Article Title: An innovative engineered IL-10 monomer strengthens T cell-mediated anti-tumor responses through anti-PD-1 cis -delivery

doi: 10.1016/j.xcrm.2025.102515

Figure Lengend Snippet: Design and in vitro characterization of anti-PD-1×IL-10M (A) CD8 + T cells were isolated from PBMCs and induced by CD3/CD28 magnetic beads for 5 days in vitro , as the exhausted CD8 + T cells. The expression of PD-1, TIM-3, or IL-10R was detected by FCM analysis. (B) Schematic diagram of anti-PD-1×IL-10M. IL-10M is linked to the C terminus of the anti-PD-1 antibody light chain via (G4S)4G. (C) Binding of each indicated molecule to CHO-human PD-1 (left) or CHO-human IL-10R cells (right) was analyzed by FCM analysis. MFI means mean fluorescence intensity. Data are representative of three independent experiments. (D) The bioactivity of the indicated proteins was detected in the reporter gene system. (E) IFN-γ secretion in the supernatant was detected by ELISA in SEB (left) or MLR (right) experiments. Statistical analyses were performed by one-way ANOVA with Dunnett’s multiple comparisons tests (ns, not significant; ∗∗∗ p ≤ 0.001; ∗∗∗∗ p ≤ 0.0001). Data are representative of two independent experiments performed on different donors. (F) The Fc region is a hinge-stabilized hIgG1 mutant subtype. Binding of each indicated molecule to C1q was analyzed by ELISA (left), and ADCC activity was detected by reporter gene assay (right). Statistical analyses were performed by two-way ANOVA with Dunnett’s multiple comparisons tests (∗∗∗∗ p ≤ 0.0001). Results are reported as the mean ± SEM.

Article Snippet: CD8 (TIL) MicroBeads, mouse , Miltenyi Biotec , Cat#: 130-116-478.

Techniques: In Vitro, Isolation, Magnetic Beads, Expressing, Binding Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Mutagenesis, Activity Assay, Reporter Gene Assay

Journal: Cell Reports Medicine

Article Title: An innovative engineered IL-10 monomer strengthens T cell-mediated anti-tumor responses through anti-PD-1 cis -delivery

doi: 10.1016/j.xcrm.2025.102515

Figure Lengend Snippet: Anti-PD-1×IL-10M acquires enhanced bioactivity through anti-PD-1-mediated cis -binding (A) Binding of each indicated molecule to HEK-IL-10R-hPD-1 reporter cells was detected by FCM analysis. MFI means mean fluorescence intensity. Data are representative of three independent experiments. (B) The bioactivity of the indicated proteins was detected using HEK-IL-10R reporter cells (left) or HEK-IL-10R-hPD-1 reporter cells (right). For PD-1 blockade, 10 nM anti-PD-1 antibody was added. Data are representative of three independent experiments. (C) Binding of each indicated molecule to exhausted CD8 + T cells induced in vitro . Data are representative of three independent experiments performed on different donors. (D) The bioactivity of the indicated proteins was detected using exhausted CD8 + T cells; 10 nM anti-PD-1 antibody was added for PD-1 blockade. Data are representative of two independent experiments performed on different donors. (E) Exhausted CD8 + T cells were induced by CD3/CD28 magnetic beads for 5 days in vitro , then incubated with indicated proteins or an IL-10R inhibitor for 4 days. The apoptosis of exhausted CD8 + T cells was detected by FCM analysis (left), and IFN-γ in the supernatant was detected by ELISA (right). Data are representative of two independent experiments performed on different donors. (F) Schematic diagram of the killing of PANC.05.04 cells by CMV-induced exhausted CD8 + T cells. CMV-induced exhausted CD8 + T cells were co-cultured with PANC pre-loaded with pp65. The PANC.05.04 tumor cell killing efficiency was detected by FCM analysis. Data are representative of three independent experiments. Results are reported as mean ± SEM. Statistical analyses were performed by paired Student’s t tests. (ns, not significant; ∗ p ≤ 0.05, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001).

Article Snippet: CD8 (TIL) MicroBeads, mouse , Miltenyi Biotec , Cat#: 130-116-478.

Techniques: Binding Assay, Fluorescence, In Vitro, Magnetic Beads, Incubation, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cell Reports Medicine

Article Title: An innovative engineered IL-10 monomer strengthens T cell-mediated anti-tumor responses through anti-PD-1 cis -delivery

doi: 10.1016/j.xcrm.2025.102515

Figure Lengend Snippet: Anti-PD-1×IL-10M exerts anti-tumor efficacy dependent on intratumoral CD8 + T cells (A and B) Anti-mPD-1×IL-10M and Isotype×IL-10M fusion proteins were radiolabeled with zirconium-89 ( 89 Zr) and then intraperitoneally injected into MC38 tumor-bearing mice. PET imaging (left) and tumor-to-muscle uptake ratio (right) were performed at different time points to observe tumor uptake (A). Biodistribution of anti-mPD-1×IL-10M and Isotype×IL-10M in different tissues (B). Statistical analyses were performed by unpaired Student’s t tests (∗ p ≤ 0.05, ∗∗ p ≤ 0.01). (C) Balb/c mice were inoculated with 1 × 10 5 CT26 tumor cells. Tumor-bearing mice ( n = 8/group) were intraperitoneally treated with indicated proteins on days 9, 12, and 15. The tumor volume of mice was measured as indicated. Statistical analyses were performed by two-way ANOVA with Dunnett’s multiple comparisons test (∗ p ≤ 0.05; ∗∗∗∗ p ≤ 0.0001). (D) The frequency of intratumoral mCD45 + , mCD3 + CD8 + T, mCD3 + CD4 + T, and mCD4 + mCD25 + FOXP3 + Treg cells in the CT26 tumors after treatment, detected by FCM analysis. Statistical analyses were performed by one-way ANOVA with Dunnett’s multiple comparisons test (∗ p ≤ 0.05; ∗∗ p ≤ 0.01). (E) C57BL/6 mice were inoculated with 1 × 10 6 MC38 tumor cells. Tumor-bearing mice ( n = 8/group) were intraperitoneally treated with indicated proteins on days 9, 13, and 16. The tumor volume of mice was measured as indicated. Statistical analyses were performed by two-way ANOVA with Dunnett’s multiple comparisons test (∗∗∗ p ≤ 0.001; ∗∗∗∗ p ≤ 0.0001). (F) The frequency of IFN-γ + GZMB + CD8 + T cells, terminally exhausted PD-1 high TOX high CD8 + T, and IFN-γ + GZMB + terminally exhausted CD8 + T cells in the MC38 tumors after anti-mPD-1×IL-10M and anti-mPD-1 antibody treatment, detected by FCM analysis. Statistical analyses were performed by one-way ANOVA with Dunnett’s multiple comparisons test (ns, not significant; ∗ p ≤ 0.05; ∗∗∗ p ≤ 0.001; ∗∗∗∗ p ≤ 0.0001). (G) C57BL/6 mice were inoculated with 5 × 10 5 MC38 tumor cells. UMAP showing the CD8 + T cell clusters of CD45 + cells treated with indicated proteins at equimolar concentrations. (H) Percentage of CD8 + T cell clusters treated with indicated proteins. (I) C57BL/6 mice were inoculated with 3 × 10 5 MC38 tumor cells. Tumor-bearing mice ( n = 6/group) were intraperitoneally treated with indicated proteins on days 8, 12, and 15. For cell depletion, mice were injected with 200 μg αCD4, αCD8, αNK, or αMo antibody 1 day before treatment and once every 3 days for three doses. The tumor growth curve of mice was monitored. Statistical analyses were performed by two-way ANOVA with Dunnett’s multiple comparisons test (∗∗∗∗ p ≤ 0.0001). Results are reported as mean ± SEM.

Article Snippet: CD8 (TIL) MicroBeads, mouse , Miltenyi Biotec , Cat#: 130-116-478.

Techniques: Injection, Imaging

Journal: Cell Reports Medicine

Article Title: An innovative engineered IL-10 monomer strengthens T cell-mediated anti-tumor responses through anti-PD-1 cis -delivery

doi: 10.1016/j.xcrm.2025.102515

Figure Lengend Snippet: Anti-PD-1×IL-10M demonstrates favorable safety both in CRS study and GLP study (A) PBMCs were isolated from three healthy human donors and incubated with negative control (PBS), positive control (LPS, anti-CD3/anti-CD28), or a series of concentrations of anti-PD-1×IL-10M and Isotype for 24 h in vitro . The secretion of IL-2, IL-4, IL-6, IL-8, IL-12p70, IFN-γ, TNF-α, and IL-1β in the supernatant was detected by LEGENDplex TM kit (BioLegend). (B) Schematic diagram of the 4-week repeated-dose toxicity study in cynomolgus monkeys. A total of 40 cynomolgus monkeys ( n = 20/sex) were randomly assigned into four groups and received anti-PD-1×IL-10M once weekly at doses of 0 (vehicle), 0.5, 2.5, or 10 mg/kg by intravenous infusion for five doses. (C) Anti-PD-1×IL-10M plasma concentration plotted up to 168 h after the first dose. (D and E) Secretory levels of IL-6 and IL-18 cytokines (pg/mL) from animal plasma at the indicated study time points. Concentrations below the lower limit of quantification (LLOQ) are reported as 0 pg/mL. (F) Male and female animal weights (kg) from day 6 to day 28 of anti-PD-1×IL-10M dosing in cynomolgus monkeys. (G) Hematology analyses show the concentration of immune cells on day 30. Reported are WBCs, RBCs, and platelets. (H) Changes in the number of reticulocytes (Retic) at the indicated study time points. (I) Peripheral blood safety markers on day 30. Reported are alanine transaminase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), hemoglobin (HGB), and monocyte. (J–M) Blood was collected at the indicated times to determine immune cell subset frequencies. Subsets reported are CD8 + T (J), CD4 + T (K), Treg (L), and NK cells (M). Statistical analyses were performed by one-way ANOVA with Dunnett’s multiple comparisons test (ns, not significant). Results are reported as mean ± SEM. LLOQ is indicated on several plots.

Article Snippet: CD8 (TIL) MicroBeads, mouse , Miltenyi Biotec , Cat#: 130-116-478.

Techniques: Isolation, Incubation, Negative Control, Positive Control, In Vitro, Clinical Proteomics, Concentration Assay