il 12 levels  (Shanghai Korain Biotech Co Ltd)

Journal: Science advances

Article Title: Multi-armored allogeneic MUC1 CAR T cells enhance efficacy and safety in triple-negative breast cancer.

doi: 10.1126/sciadv.adn9857

Figure Lengend Snippet: Fig. 4. TGFBR2KO armored UCARTM1 shows resistance to inhibitory effects of TGFB1. (A) Schematic representation of the non-alloreactive CAR T cell with TRAC and TGFBR2 knockout with TALEN (UCARTM1∆TGFBR2). (B) Functional phenotyping of TGFBR2 knockout edited UCARTM1 compared to the unedited UCARTM1 via flow cytometry analysis of pSMAD2/3 staining in the presence of TGFB1. (C) Flow cytometry analysis of the percentage of CD25+ cells present in UCARTM1 and UCARTM1∆TGFBR2 following activation with MUC1 recombinant protein, in the presence or absence of TGFB1 (n = 2). Statistical significance was calculated using unpaired t test. (D) Proliferation assay for MUC1 recombinant protein activated UCARTM1 and UCARTM1∆TGFBR2 in the presence or absence of TGFB1 at days 0, 4, 6, 8, and 11 (n = 2 technical replicates per time point). Statistical significance was determined using a two-way ANOVA. (E) Design of in vivo experiment and individual tumor growth comparison of tumors treated intravenously with 5 million UCARTM1∆TGFBR2, UCARTM1, NTD, or PBS (n = 4 to 5 per cohort) for donor 1. Experiment repeated with donor 2 is shown in the Supplementary Materials. Two- way ANOVA was performed for comparisons over time for tumor growth. (F) Design of in vivo experiment, individual tumor growth comparison of tumors, and (G) Kaplan- Meier survival analysis of cohorts treated intratumorally with 2 million UCARTM1∆TGFBR2 (n = 4), UCARTM1 (n = 4), NTD (n = 2), or PBS (n = 3). Each point represents a biological replicate for (E) to (G). Statistical significance was calculated using mixed-effects analysis comparisons over time for tumor growth and the log-rank Mantel-Cox test. *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns (not significant) indicates P > 0.05. (H) TGFB1 ELISA analysis of HCC70-GFP tumors averaging 50 to 100, 250, 450, and 600 mm3 per mm3 of tumor (n = 2 to 6). Each point represents a biological replicate. Treatments were done using 2 × 106 or 5 × 106 CAR+ cells as indicated on each in vivo design.

Article Snippet: IL- 12, IFNG, and TGFB1 ELISAs IL- 12 levels were measured with Quantikine ELISA Human IL- 12 p70 Immunoassay (R&D Systems, no. D1200) using 30 μl of blood.

Techniques: Knock-Out, Functional Assay, Flow Cytometry, Staining, Activation Assay, Recombinant, Proliferation Assay, In Vivo, Comparison, Enzyme-linked Immunosorbent Assay

Journal: Science advances

Article Title: Multi-armored allogeneic MUC1 CAR T cells enhance efficacy and safety in triple-negative breast cancer.

doi: 10.1126/sciadv.adn9857

Figure Lengend Snippet: Fig. 5. PD1KO/IL-12KI and TGFBR2KO armored CAR T cells clear tumors within weeks without relapse with less CAR T cell expansion. (A) Schematic representation of the non- alloreactive CAR T cell with TRAC, PDCD1, and TGFBR2 knockouts and IL-12 knock-in. (B) Proliferation assay of MUC1 recombinant protein–activated UCARTM1∆PD1/IL12 and UCARTM1∆PD1/IL12; ∆TGFBR2 with or without TGFB1 (n = 2 technical replicates per time point). Two-way ANOVA was performed for statistical significance over time. (C) Cytotoxic assay of T47D cells cocultured with NTD, UCARTM1, UCARTM1∆TGFBR2, UCARTM1∆PD1/IL12, and UCARTM1∆PD1/IL12; ∆TGFBR2in the presence of TGFB1 with a 10-to-1 E:T ratio. CAR T cells were rechallenged after 24 hours (n = 3). Each point represents a technical replicate. Statistical significance was calculated using unpaired t test. (D) Design of in vivo experiment. (E) Tumor growth curve for cohorts intravenously treated with 5 million UCARTM1∆PD1/IL12; ∆TGFBR2, UCARTM1∆PD1/IL12, UCARTM1∆TGFBR2, UCARTM1, NTD, or PBS (n = 5 to 6 per cohort) and (F) flow cytometry analysis of PB collected from all cohorts, 40 days after treatment for number and percent of hCD45+ cells. Two-way ANOVA was performed for comparisons for tumor growth, and ordinary one-way ANOVA was performed to determine the statistical significance for (F). (G) Color mapping of tumor clearance and inflammation in the mam- mary glands of UCARTM1∆PD1/IL12- and UCARTM1∆PD1/IL12; ∆TGFBR2-treated cohorts (n = 5 and 6, consecutively) and representative dissection images of tumor cell injected (inj.) and no tumor cell injected mammary fat pad (MFP) (Control; Ctrl) of one animal each. (H) Kaplan-Meier survival analysis of cohorts treated with UCARTM1∆PD1/IL12; ∆TGFBR2, UCARTM1∆PD1/IL12, UCARTM1∆TGFBR2, UCARTM1, NTD, or PBS (n = 4 to 6 per cohort). Each point represents a biological replicate for (E) to (G). Statistical significance was calculated using the log-rank Mantel-Cox test in (H). *P < 0.05, **P ≤ 0.01, ****P ≤ 0.000, ns (not significant) indicates P > 0.05. All treatments were done using 5 × 106 CAR+ cells.

Article Snippet: IL- 12, IFNG, and TGFB1 ELISAs IL- 12 levels were measured with Quantikine ELISA Human IL- 12 p70 Immunoassay (R&D Systems, no. D1200) using 30 μl of blood.

Techniques: Knock-In, Proliferation Assay, Recombinant, In Vivo, Flow Cytometry, Dissection, Injection, Control

Journal: International Journal of Nanomedicine

Article Title: IL-12-Overexpressed Nanoparticles Suppress the Proliferation of Melanoma Through Inducing ICD and Activating DC, CD8+ T, and CD4+ T Cells

doi: 10.2147/ijn.s442446

Figure Lengend Snippet: Figure 2 CPP transferred IL-12 into B16F10 cells and CPP-IL-12 induced immunogenic cell death. (A) The structure of IL-12 vector. (B) Western blot detection of IL-12 expression. **P < 0.01, Student’s t-test. (C) ELISA. CPP-IL-12 increased IL-12 levels in the supernatant of B16-F10 cells, and 2.0 W/cm2 of laser further increased IL-12 levels. **P < 0.01, ANOVA. (D) Live/dead staining assay. Scale bars=100 μm. (E) The number of dead cells. CPP-IL-12 increased and 2.0 W/cm2 of laser further increased the number of dead cells. **P < 0.01, ANOVA. (F) Cell apoptosis analysis. CPP-IL-12 increased and laser treatment further increased the number of apoptotic cells. (G) Immunofluorescence. Green color, CALR expression. Scale bars=2 μm. (H, I) ELISA analysis of extracellular and intracellular HMGB1 levels, respectively. *P < 0.05, **P < 0.01, ANOVA. (J) ATP levels. The ATP levels decreased in CPP-IL-12-treated cells, and laser treatment further reduced ATP content. **P < 0.01, ANOVA. (K) CPP increased CD80+/CD86+ expression of DC.

Article Snippet: IL-12 and HMGB1 levels were estimated using ELISA kits (CSB-E04600m and CSB-E08225M, CUSABIO, Wuhan, China) in accordance with the manufacturer′s instructions.

Techniques: Plasmid Preparation, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence

Journal: Journal of Nanobiotechnology

Article Title: Cu-doped TiO 2 nanoparticles improve local antitumor immune activation and optimize dendritic cell vaccine strategies

doi: 10.1186/s12951-023-01844-z

Figure Lengend Snippet: A Histogram displaying the ratio of CD8 + T cells over CD4 + T cells obtained upon in vitro stimulation by OVA-peptide loaded DCs that had either been left untreated (control) or were activated classically, or upon exposure to 33% Cu-doped TiO 2 NPs. B , C Histograms displaying the relative level (control: 100%) of B) granzyme B release, or C) perforin release from T cells obtained from part A that had been exposed to the OVA-derived SIINFEKL peptide. D , E Histograms displaying the level of D) IL12p70 release or E) relative Ccr7 surface expression (control: 100%) on DCs that had either been left untreated (control) or were activated classically, or exposed to 33% Cu-doped TiO 2 NPs. F , G) Histograms displaying the F) number of migrated DCs or G) the level of IL12p70 secreted from migratory DCs obtained from DCs that had either been left untreated (control) or were activated classically, or exposed to 33% Cu-doped TiO 2 NPs and subsequently exposed to the CCR7 ligand 6C-kine. H , I Histograms displaying the in vivo antibody titer for H) OVA-specific IgG1 or I) OVA-specific IgG2α obtained upon intravenous administration of OVA-pulsed DCs that had either been left untreated (control) or were activated classically, or exposed to 33% Cu-doped TiO 2 NPs. J Histogram displaying the level of IFNγ obtained from ex vivo splenocytes obtained from animals receiving the different types of OVA-pulsed DCs and incubated with OVA ex vivo. K Tumor volumes obtained from OVA-expressing E0771 tumors grafted subcutaneously in C57Bl6 mice receiving the different OVA-pulsed DCs that had either been left untreated (control) or were activated classically, or exposed to 33% Cu-doped TiO 2 NPs. L , M Histograms displaying the relative level (control: 100%) of L) granzyme B release, or M) perforin release from CD8 + T cells isolated from OVA-E0771 tumor-bearing animals having received OVA-pulsed DC grafts of the different DC types and then exposed to the OVA-derived SIINFEKL peptide ex vivo. N Histogram displaying the level of OVA-specific CD8 + T cells isolated from the spleen of OVA-E0771 tumor-bearing animals having received OVA-pulsed DC grafts of the different DC types

Article Snippet: Media were then removed and fresh media (5 ml) was given with either no additions (controls), 33% Cu-doped TiO 2 NPs at 40 μg/ml (NP condition) or addition of maturation factors IL1β (25 ng/ml) TNFα (50 ng/ml) and IFNγ (1000 units/ml) (all from PeproTech; this is the “classical” activation scheme) for 24 h. Media were then removed and cells were incubated with soluble recombinant CD40L (BioTechne) at 16 μg/ml for 24 h after which the supernatants was collected and used to determine IL12p70 levels by ELISA (M1270, R&D Systems).

Techniques: In Vitro, Control, Derivative Assay, Expressing, In Vivo, Ex Vivo, Incubation, Isolation