Journal: Experimental and therapeutic medicine

Article Title: PHLDA3 inhibition attenuates endoplasmic reticulum stress-induced apoptosis in myocardial hypoxia/reoxygenation injury by activating the PI3K/AKT signaling pathway.

doi: 10.3892/etm.2021.10045

Figure Lengend Snippet: Figure 4. Blockage of the p‑PI3K/AKT pathway inhibits the protective effects of PHLDA3 inhibition following H/R injury. (A) Cell Counting Kit‑8 assays were used to evaluate cell viability. (B) LDH release was detected by ELISA. (C) Apoptotic rate in each group. mRNA levels of ERS markers, including (D) CHOP, (E) GRP78 and (F) caspase‑12, were measured by reverse transcription‑quantitative PCR. Data are presented as the mean ± standard deviation. n=4/group. *P<0.05 vs. H/R + AdshPHLDA3 + PBS. #P<0.05 vs. H/R + AdshRNA + PBS. PHLDA3, pleckstrin homology‑like domain family A member 3; H/R, hypoxia/ reoxygenation; LDF, lactate dehydrogenase; GRP78, 78 kDa glucose‑regulated protein; AdshRNA, adenovirus encoding scrambled short hairpin RNA; AdshPHLDA3, adenoviral vectors encoding PHLDA3 shRNA; NS, not significant.

Article Snippet: Antibodies against p‐AKT (cat. no. 4060; 1:600), AKT (cat. no. 4691; 1:400) and CHOP (cat. no. 5554; 1:600) were purchased from Cell Signaling Technology, Inc. Horseradish peroxidase‐conjugated goat‐anti rabbit secondary antibodies were obtained from BIOSS.

Techniques: Inhibition, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, shRNA

Journal: The FASEB Journal

Article Title: CD30 plays a role in T‐dependent immune response and T cell proliferation

doi: 10.1096/fj.202301747rr

Figure Lengend Snippet: FIGURE 1 Expression of CD30 in the T cells and B cells and generation of CD30-deficient mice. (A) The indicated hematopoietic cells were sorted from wild-type mice. The level of CD30 mRNA expression was measured by qRT-PCR, using β-Actin as internal control. (B) The frequency of CD30+ T cells in CD4 T cells upon TCR stimulation (left). The overlays show CD30 surface expression of CD4 T cells upon TCR stimulation (right). (C) The frequency of CD30+ T cells in CD8 T cells upon TCR stimulation (left). The overlays show CD30 surface expression of CD8 T upon TCR stimulation (right). (D) CD30 expression level was detected by qRT-PCR. (E) Western blotting analysis of CD30 expression in splenic T cells. Purified splenic T cells were stimulated for 48 h on tissue culture plate coated with anti-CD3 (10 μg/mL) plus anti-CD28 (5 μg/mL) and IL-2 (100 ng/mL). The cell lysates were subjected to direct Western blot analysis with the indicated antibodies. Data shown are representative of or obtained from 3 (A–E) independent experiments. Mean ± SD is shown.

Article Snippet: Rabbit monoclonal anti- CD30 (#54535) was purchased from Cell Signaling Technology.

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Purification

Journal: The FASEB Journal

Article Title: CD30 plays a role in T‐dependent immune response and T cell proliferation

doi: 10.1096/fj.202301747rr

Figure Lengend Snippet: FIGURE 3 Serum immunoglobulin levels and serum antibody response to TD antigens in CD30-KO and wild-type mice. (A) Serum immunoglobulin isotype levels in wild-type and CD30-KO mice were measured by ELISA. (B) Serum NP-specific immunoglobulin isotype response to the TD antigen NP-KLH immunization in wild-type and CD30-KO mice. Sera were collected at the indicated time points after immunization and antigen-specific antibodies were measured by ELISA. (C) CD30-KO and wild-type mice immunized with NP-KLH. Splenocytes from CD30-KO and wild-type mice were stained with anti-B220, anti-CD95, and anti-GL7 at day 9 after immunization and analyzed by FACS. Percentages indicate cells in the gated B220+ cells. (D) Dot plots show percentages and numbers of GC B cells in the spleen of CD30-KO and wild-type mice. (E) Splenic mature B cells were stimulated with LPS for 48 h, and cell lysates were subjected to direct Western blot analysis with the indicated antibodies. (F) The intensities of protein bands were quantitated using Image-J software. Data shown are representative of or obtained from 6 (A), 4 (B, E, F), 5 (C, D), or 4 (E, F) CD30-KO and wild-type littermates. Mean ± SD is shown. *p < .05, **p < .01.

Article Snippet: Rabbit monoclonal anti- CD30 (#54535) was purchased from Cell Signaling Technology.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Software

Journal: The FASEB Journal

Article Title: CD30 plays a role in T‐dependent immune response and T cell proliferation

doi: 10.1096/fj.202301747rr

Figure Lengend Snippet: FIGURE 4 CD30 deficiency has no effect on T-cell development. (A) Dot plots show numbers of total thymocytes in CD30-KO and wild- type mice. (B) Thymocytes from CD30-KO and wild-type mice were stained with anti-CD4 and anti-CD8 and analyzed by FACS. Percentages indicate cells in the gated live cells. (C) Dot plots show percentages and numbers of DN, DP, CD4, and CD8 T cells in the thymocytes of CD30-KO and wild-type mice. (D) Thymocytes from CD30-KO and wild-type mice were stained with anti-CD4, anti-CD8, anti-CD25, and anti-CD44 and analyzed by FACS. Percentages indicate cells in the gated DN T cells. (E) Dot plots show percentages and numbers of DN1, DN2, DN3, and DN4 T cells in the DN T cells of CD30-KO and wild-type mice. (F) Splenocytes from CD30-KO and wild-type mice were stained with anti-CD4 and anti-CD8 and analyzed by FACS. Percentages indicate cells in the gated live cells. (G) Dot plots show percentages and numbers of CD4 and CD8 T cells in the spleen of CD30-KO and wild-type mice. Data shown are representative of or obtained from 10 (A–G) CD30-KO and wild-type littermates. Mean ± SD is shown.

Article Snippet: Rabbit monoclonal anti- CD30 (#54535) was purchased from Cell Signaling Technology.

Techniques: Staining

Journal: The FASEB Journal

Article Title: CD30 plays a role in T‐dependent immune response and T cell proliferation

doi: 10.1096/fj.202301747rr

Figure Lengend Snippet: FIGURE 5 CD30 deficiency reduces TCR-mediated proliferation. (A) Splenic T cells were labeled with CFSE, according to the manufacturer's recommendation, and stimulated as indicated for 72 h before analysis for cell proliferation (left). Dot plots show the percentages of proliferate rate (right). (B) Splenic T cells were stimulated as indicated for 72 h, stained with PI, and analyzed for cell cycle profile by FACS (left). Dot plots show percentages of cells in S/G2/M phase after stimulation (right). Data shown are representative of or obtained from 3 (A, B) independent experiments. Mean ± SD is shown. *p < .05, ** p < .01.

Article Snippet: Rabbit monoclonal anti- CD30 (#54535) was purchased from Cell Signaling Technology.

Techniques: Labeling, Staining

Journal: The FASEB Journal

Article Title: CD30 plays a role in T‐dependent immune response and T cell proliferation

doi: 10.1096/fj.202301747rr

Figure Lengend Snippet: FIGURE 6 CD30 deficiency reduces TCR-mediated T-cell activation. (A) Total thymocytes were stimulated with anti-CD3 (10 μg/ mL) for the indicated time and cell lysates were subjected to direct Western blot analysis with the indicated antibodies. (B) The intensities of protein bands were quantitated using Image-J software. (C, D) Splenic T cells from CD30-KO and wild-type littermates were stimulated with anti-CD3 for 72 h and analyzed by FACS and ELISA. (C) Splenic T cells were stained with anti-CD4, anti-CD8, anti-CD25, and anti- CD69 upon TCR stimulation. Dot plots show percentages of cells in CD4 and CD8 T cells after stimulation. (D) ELISA analysis of IL-6 and IFN-γ released in the splenic T cell culture supernatant. Data shown are representative of or obtained from 6 (A, B) or 3 (C, D) independent experiments. Mean ± SD is shown. *p < .05.

Article Snippet: Rabbit monoclonal anti- CD30 (#54535) was purchased from Cell Signaling Technology.

Techniques: Activation Assay, Western Blot, Software, Enzyme-linked Immunosorbent Assay, Staining, Cell Culture

Journal: The FASEB Journal

Article Title: CD30 plays a role in T‐dependent immune response and T cell proliferation

doi: 10.1096/fj.202301747rr

Figure Lengend Snippet: FIGURE 7 Analysis of CD30-KO gene expression signature in TCR-mediated T cells. (A, B) Splenic T cells from CD30-KO and wild- type littermates were sorted and stimulated with anti-CD3/anti-CD28/IL-2 for 48 h and subjected to high-throughput RNA-sequencing. (A) Volcano plot of differentially expressed genes between CD30-KO and wild-type T cells. (B) Significantly downregulation pathways from the KEGG pathway database in CD30-KO and wild-type T cells. (C) The relative mRNA levels of LC3 and Atg7 were quantified by qRT-PCR in CD30-KO and wild-type T cells with anti-CD3/anti-CD28/IL-2 stimulation. The relative mRNA levels of the indicated genes in CD30-KO and wild-type T cells were normalized to β-Actin. (D) Splenic T cells were stimulation with anti-CD3/anti-CD28/IL-2 and cell lysates were subjected to direct Western blot analysis with the indicated antibodies (left). The intensities of protein bands were quantitated using Image-J software (right). Data shown are representative of or obtained from 2 (A, B) or 3 (C, D) independent experiments. Mean ± SD is shown. *p < .05, **p < .01.

Article Snippet: Rabbit monoclonal anti- CD30 (#54535) was purchased from Cell Signaling Technology.

Techniques: Gene Expression, High Throughput Screening Assay, RNA Sequencing, Quantitative RT-PCR, Western Blot, Software

Journal: Molecular Therapy Oncolytics

Article Title: Nanobody-derived bispecific CAR-T cell therapy enhances the anti-tumor efficacy of T cell lymphoma treatment

doi: 10.1016/j.omto.2023.07.007

Figure Lengend Snippet: Construction of the Nb phage library and screening of anti-CD30 Nbs and anti-CD5 Nbs (A) Schematic of construction of the Nb phage library and screening of Nbs. (B) The variable domain of heavy chain of heavy-chain antibody (VHH) genes were obtained by two-step PCR, and the library size was measured by counting the colonies after serial dilution (M, DNA marker; 1, PCR product). (C) Monoclonal phage ELISA. 24 positive phages from the three rounds of panning were tested for binding affinity by ELISA with CD30- and CD5-coated plates. (D) Flow cytometry screening of monoclonal phages capable of binding to cells. Karpas-299 cells were used as target cells to validate positive monoclonal phages by flow cytometry. Negative control (NC) phages that did not express Nbs were used as a negative control.

Article Snippet: Expression of CD30 antigen was detected by an anti-human CD30 antibody (rabbit anti-CD30 polyclonal antibody, Bioss, bs-2495R).

Techniques: Serial Dilution, Marker, Enzyme-linked Immunosorbent Assay, Binding Assay, Flow Cytometry, Negative Control

Journal: Molecular Therapy Oncolytics

Article Title: Nanobody-derived bispecific CAR-T cell therapy enhances the anti-tumor efficacy of T cell lymphoma treatment

doi: 10.1016/j.omto.2023.07.007

Figure Lengend Snippet: In vitro binding ability validation of the screened Nbs (A) The biding affinity of Nb-Fc to CD30 or CD5 was measured by ELISA. (B) Three anti-CD30 Nbs and four anti-CD5 Nbs were selected, and we performed ELISA at the indicated dilution ratios by incubation with the corresponding antigens. Fc served as negative control. (C) Detection of the binding affinity of the Nb-Fc or scFv-Fc antibodies to CD5 or CD30, respectively, by ELISA. (D) Single-target NbCD30 or NbCD5-CAR-T cells were co-cultured with Karpas-299 cells (CD30 + CD5 + ) at different effector-to-target ratios for 24 h, and the specific cytotoxicity was determined by Lactic Dehydrogenase (LDH) assay. ∗∗∗∗p < 0.0001 for one-way ANOVA with multiple-comparisons test. All data are mean ± standard error.

Article Snippet: Expression of CD30 antigen was detected by an anti-human CD30 antibody (rabbit anti-CD30 polyclonal antibody, Bioss, bs-2495R).

Techniques: In Vitro, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Negative Control, Cell Culture, Lactate Dehydrogenase Assay

Journal: Molecular Therapy Oncolytics

Article Title: Nanobody-derived bispecific CAR-T cell therapy enhances the anti-tumor efficacy of T cell lymphoma treatment

doi: 10.1016/j.omto.2023.07.007

Figure Lengend Snippet: The binding ability was predicted by molecular docking and verified by SPR experiments (A) Molecular docking prediction model of anti-CD30/CD5 Nbs or scFv to corresponding antigens. The light blue structure indicates the antigen, the dark blue structure indicates the antibody, the interface area (red) indicates the region of the two proteins in contact with each other, and ΔG indicates the free energy of binding. (B) SPR experiments were performed to measure the binding affinity of Nb-Fc or scFv-Fc on its corresponding antigen.

Article Snippet: Expression of CD30 antigen was detected by an anti-human CD30 antibody (rabbit anti-CD30 polyclonal antibody, Bioss, bs-2495R).

Techniques: Binding Assay

Journal: Molecular Therapy Oncolytics

Article Title: Nanobody-derived bispecific CAR-T cell therapy enhances the anti-tumor efficacy of T cell lymphoma treatment

doi: 10.1016/j.omto.2023.07.007

Figure Lengend Snippet: Construction of bispecific Nb/scFv-derived CAR-T cells to verify in vitro function (A) Schematic of CAR structure and single- or bispecific scFv-CAR. (B) Schematic of single NbCD30/NbCD5-CAR or bispecific NbCD30-CD5-CAR. (C) Single-or bispecific scFv-CAR-T cells were co-cultured with Karpas-299 cells (CD30 + CD5 + ) or SupT1 cells (CD30 + CD5 − ) at the indicated E:T ratios for 24 h, and the specific cytotoxicity was determined by LDH assay. The data shown are representative results of three independent replication experiments. (D) Single-or bispecific Nb-CAR-T cells were co-cultured with Karpas-299 cells (CD30 + CD5 + ) or SupT1 cells (CD30 + CD5 − ) at different E:T ratios for 24 h, and the specific cytotoxicity was determined by LDH assay. The data shown are representative results of three independent replication experiments. (E) Bispecific Nb-CAR-T and scFv-CAR-T cells were co-cultured with Karpas-299 cells (CD30 + CD5 + ) or Raji cells (CD30 − CD5 − ) at different E:T ratios for 24 h, and the specific cytotoxicity was determined by LDH assay. (F) NbCD30/CD5-CAR-T cells or mock T cells (untransduced T cells) were incubated with tumor target cells (Karpas-299) for 24 h. The levels of IFN-γ, TNF-α, IL-2, and granzyme B in culture supernatants were measured by ELISA. (G) CAR-T cells or control T cells were incubated with tumor target cells (Karpas-299) at a 1:1 ratio for 24 h. Secretion of IFN-γ was measured by ELISpot assay. All data are representative of three independent replication experiments. All data are mean ± standard error; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 for one-way ANOVA.

Article Snippet: Expression of CD30 antigen was detected by an anti-human CD30 antibody (rabbit anti-CD30 polyclonal antibody, Bioss, bs-2495R).

Techniques: Derivative Assay, In Vitro, Cell Culture, Lactate Dehydrogenase Assay, Incubation, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Molecular Therapy Oncolytics

Article Title: Nanobody-derived bispecific CAR-T cell therapy enhances the anti-tumor efficacy of T cell lymphoma treatment

doi: 10.1016/j.omto.2023.07.007

Figure Lengend Snippet: Bispecific CD30-CD5-CAR-T cells are more effective for tumor growth suppression (A and B) 6 × 10 6 Karpas-299 cells were inoculated subcutaneously into NCG mice. The tumor reached 100 mm 3 after 7–10 days. Various T cells were infused via tail vein injection. Tumor engraftment was monitored every 2–3 days (1–2 μg of human IL-2 per mouse was injected at a frequency of 2–3 days). s.c., subcutaneous; i.v., intravenous; i.p., intraperitoneal. Tumor volumes were monitored after injection of CAR-T cells (scale bar, 10 mm). (C) The tumor mass was quantified. All data are mean ± standard error; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 for one-way ANOVA.

Article Snippet: Expression of CD30 antigen was detected by an anti-human CD30 antibody (rabbit anti-CD30 polyclonal antibody, Bioss, bs-2495R).

Techniques: Injection

Journal: Molecular Therapy Oncolytics

Article Title: Nanobody-derived bispecific CAR-T cell therapy enhances the anti-tumor efficacy of T cell lymphoma treatment

doi: 10.1016/j.omto.2023.07.007

Figure Lengend Snippet: Nb-derived CD30 and CD5 bispecific CAR-T cells enhanced anti-tumor potency in vivo (A) Fluorescence-activated cell sorting (FACS) analysis shows the proportion of tumor-infiltrating CAR-T cells and cytokine production capacity. (B) Immunofluorescence staining of tumor tissue sections. The tumor tissue sections were analyzed for immunofluorescence by staining with anti-human CD8 (red), anti-human IFN-γ (green), and anti-human granzyme B (pink) antibodies, and the nuclei were stained with DAPI (blue). The statistical plot shows the mean fluorescence intensity statistics for CD8, IFN-γ, and granzyme B expressed as the mean ± SD from three randomly selected fields of thin tumor sections. Scale bar, 20 μm. (C) Survival curve of mice after tumor inoculation and then injection of the indicated CAR-T cells. ∗p < 0.05, ∗∗p < 0.01, log rank Mantel-Cox test. All data are mean ± standard error; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, one-way ANOVA.

Article Snippet: Expression of CD30 antigen was detected by an anti-human CD30 antibody (rabbit anti-CD30 polyclonal antibody, Bioss, bs-2495R).

Techniques: Derivative Assay, In Vivo, Fluorescence, FACS, Immunofluorescence, Staining, Injection