Journal: Cell Reports Medicine

Article Title: Miltefosine reinvigorates exhausted T cells by targeting their bioenergetic state

doi: 10.1016/j.xcrm.2024.101869

Figure Lengend Snippet: Miltefosine enhances CAR-T cell efficacy against solid tumors in vivo (A) Experimental timeline of CDX model and the fold change of tumor volume in CAR-T cell-treated CDX mouse model in the presence or absence of miltefosine administration (PBS group, n = 3; other groups, n = 5). (B) Tumor weight of each group at the end of experiment (PBS group, n = 3; other groups, n = 5). (C) The percentages of mesothelin- and hCD3-positive area in tumor tissues (PBS group, n = 2; other groups, n = 5). Scale bar: 200 μm. (D) The fold change of tumor volume in CDX mouse model treated with CAR-T cells derived from another donor, in the presence or absence of miltefosine or anti-PD-1 antibody treatment ( n = 4). (E) The proportion of hCD3 + T cells in the peripheral blood of CDX mouse model ( n = 4). (F) The fold change of tumor volume in CAR-T cell-treated PDX mouse model, in the presence or absence of miltefosine or anti-PD-1 antibody treatment ( n = 4). (G) The proportion of hCD3 + T cells in the peripheral blood of PDX mouse model ( n = 4). Unpaired t test was used in statistical analysis. (H) Experimental timeline of the OT-1-treated melanoma isograft tumor model. (I) The fold change in tumor volume in the OT-1-treated melanoma isograft tumor model in the presence or absence of miltefosine administration ( n = 7). Unpaired t test was used in statistical analysis. NS, not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. All error bars denote SEM. See also Figure S7 .

Article Snippet: Rabbit anti- human CD3 Antibody (clone EP41) , ZSGB-BIO , Cat#ZA-0503.

Techniques: In Vivo, Derivative Assay

Journal: Cell Reports Medicine

Article Title: Miltefosine reinvigorates exhausted T cells by targeting their bioenergetic state

doi: 10.1016/j.xcrm.2024.101869

Figure Lengend Snippet:

Article Snippet: Rabbit anti- human CD3 Antibody (clone EP41) , ZSGB-BIO , Cat#ZA-0503.

Techniques: Functional Assay, Flow Cytometry, Control, Recombinant, Lysis, Activation Assay, Transfection, Luciferase, Viability Assay, SYBR Green Assay, RNA Sequencing Assay, Construct, Plasmid Preparation, Software

Journal: International Journal of Ophthalmology

Article Title: Association of TCR-signaling pathway with the development of lacrimal gland benign lymphoepithelial lesions

doi: 10.3980/j.issn.2222-3959.2015.04.08

Figure Lengend Snippet: Primers used for RT-PCR

Article Snippet: All control patients had normal circulating IgG4 ( ). table ft1 table-wrap mode="anchored" t5 caption a7 Groups Mean age (a) Gender Affected eyes IgG4 level M F L R Bilateral High Normal Experimental group 50 2 7 2 0 7 9 0 Control group 46.4 3 6 5 4 0 0 9 Open in a separate window caption a8 Baseline clinical characteristics of patients in the experimental and control groups Major reagents The following reagents were used: polymerase chain reaction (PCR) mix kit (Sangon Biotech, Shanghai, China); RNA reverse transcription kit (New England Biolabs, Ipswich, MA, USA); rabbit anti-human CD3 and rabbit anti-human CD4 antibodies (ZSGB-Bio, Beijing, China); mouse anti-human IL-10 antibody (Abcam, Cambridge, MA, USA); universal two-step immunohistochemistry kit, horse radish peroxidase (HRP)-conjugated goat anti-mouse IgG, and HRP-conjugated goat anti-rabbit IgG (ZSGB-Bio).

Techniques:

Journal: Experimental Animals

Article Title: NOD- Rag2 null IL-2Rγ null Mice: An Alternative to NOG Mice for Generation of Humanized Mice

doi: 10.1538/expanim.63.321

Figure Lengend Snippet: Percentages of T-cell subsets in the spleen, thymus, and peripheral blood of HSC-transplanted mice

Article Snippet: To identify human cells in the spleen of transplanted mice, the spleens were removed from the mice after blood removal, and tissues were fixed with 10 nM formalin (10 nM Mildform; Wako, Tokyo, Japan), embedded in paraffin, and stained with hematoxylin and eosin (HE), mouse anti-human CD45 antigen monoclonal antibody (clone 2B11+PD7/26; Dako Cytomation, Glostrup, Denmark), rabbit anti-human CD3 monoclonal antibody (clone SP7; Nichirei, Tokyo, Japan), and mouse anti-human CD79a monoclonal antibody (clone JCB117; Nichirei).

Techniques: Injection

Journal: Nature Communications

Article Title: Re-designing Interleukin-12 to enhance its safety and potential as an anti-tumor immunotherapeutic agent

doi: 10.1038/s41467-017-01385-8

Figure Lengend Snippet: Ad-TD-nsIL-12 is an effective treatment for subcutaneous hamster PaCa. 2 × 10 6 HPD1NR cells were seeded into the right flank of Syrian hamsters. When tumor volumes reached 300 mm 3 , seven hamsters per group were each injected i.t. with 100 µl PBS, 1 × 10 9 PFU Ad-TD-LUC or Ad-TD-nsIL-12/IL-12 on days 0, 2, 4, 6, 8, and 10. a Mean tumor sizes and SEM are shown for each group and compared using a one-way ANOVA with post hoc Tukey’s Multiple Comparison Test. * p < 0.05, *** p < 0.001. b Kaplan–Meier survival curves were generated and a log-rank (Mantel–Cox) test used to analyze significance. ** p < 0.01, *** p < 0.001. Treatment with both Ad-TD-IL-12 and Ad-TD-nsIL-12 resulted in 100% survival in this model. c Hamsters that had cleared tumors after i.t treatment with Ad-TD-nsIL-12 during efficacy experiments were re-challenged 4 weeks later in the opposite flank with 4 × 10 6 HPD1NR or 5 × 10 6 HaK cells, or were injected i.p with anti-CD3 mAb (500 μg/injection) on the day before re-challenge with HPD1NR cells. In parallel, naive hamsters were injected with 5 × 10 6 HaK cells at the same sites and tumor growth measured and analyzed using a one-way ANOVA with post hoc Tukey’s Multiple Comparison Test. *** p < 0.001

Article Snippet: Splenocytes and lymph node cells (1 × 10 6 ) were prepared and stained with mAb against Syrian hamster CD3 and mAb against mouse CD4-conjugated with FITC (GK1.5, cross-reactive with Syrian hamster, BD), followed by secondary APC-conjugated rabbit anti-mouse antibody for CD3 staining (BD).

Techniques: Injection, Generated

Journal: Nature Communications

Article Title: Re-designing Interleukin-12 to enhance its safety and potential as an anti-tumor immunotherapeutic agent

doi: 10.1038/s41467-017-01385-8

Figure Lengend Snippet: Ad-TD-nsIL-12 is an effective and safe treatment for orthotopic PaCa in Syrian hamsters. Six days after Hap-T1 cells were seeded into the tail of the pancreas, hamsters were injected i.p. with 500 µl PBS, 1 × 10 9 PFU Ad-TD-LUC, Ad-TD-IL-12 or Ad-TDnsIL-12 ( a ) or 2.5 × 10 9 PFU Ad-TD-LUC, 2.5 × 10 9 PFU Ad-TD-nsIL-12 or 5 × 10 8 PFU Ad-TD-IL-12 ( b ) ( n = 7/group) on days 0, 2, 4, 6, 8, and 10. Kaplan–Meier survival curves were generated. Significance was assessed using the log-rank (Mantel–Cox) test. * p < 0.05, ** p < 0.01. c − h Animals were treated as b ( n = 3/time point/group) and killed on day 3, 7, and 14 after last viral treatments. c Representative images of immunohistochemical staining for Hexon at day 7. Hexon-positive cells were counted in five high-power fields (HPF) from each tumor section (×200). ND: not detected. d Infectious virion recovery from tumor tissue was determined by TICD50 using JH293 cells. e Tumors, lung, and livers were analyzed by qPCR for the copy numbers of the viral E1A gene after treatment with Ad-TD-nsIL-12 at 2.5 × 10 9 PFU/injection on day 0, 2, and 4. The sensitivity of the assay is illustrated by the dotted line. Mean and SEM are shown for each group and compared using an independent t -test. * p < 0.05, ** p < 0.01. f Mean tumor volumes and SEM are shown for each group. Statistical analysis was carried out using a one-way ANOVA with post hoc Tukey’s Multiple Comparison Test. * p < 0.05. g Representative images of immunohistochemical staining for CD3 at day 7. Quantitative scores of lymphocyte infiltration within tumors are shown (right panel) from five HPF from each tumor section (×200). The scoring was conducted within the tumor and stroma and necrotic areas were avoided. The extent of positive cells was categorized into the following four grades: 1, <15 cells/HPF; 2, 16–30 cells/HPF; 3, 31–45 cells/HPF; 4, >45 cells/HPF. ND: not detected. h IL-12 expression in sera was detected by ELISA. i Representative histopathology of HE staining of livers after three i.p injections of virus into hamsters bearing orthotopic PaCa tumors ( n = 3/group) on day 0, 2, and 4, using the same dose as in b . Livers were collected one day following the last injection and analyzed using HE staining (×200)

Article Snippet: Splenocytes and lymph node cells (1 × 10 6 ) were prepared and stained with mAb against Syrian hamster CD3 and mAb against mouse CD4-conjugated with FITC (GK1.5, cross-reactive with Syrian hamster, BD), followed by secondary APC-conjugated rabbit anti-mouse antibody for CD3 staining (BD).

Techniques: Injection, Generated, Immunohistochemical staining, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Histopathology

Journal: Nature Communications

Article Title: Re-designing Interleukin-12 to enhance its safety and potential as an anti-tumor immunotherapeutic agent

doi: 10.1038/s41467-017-01385-8

Figure Lengend Snippet: Ad-TD-nsIL-12 efficacy is dependent on hamster CD3+/CD4− immune cell subsets. a , b Syrian hamsters were inoculated subcutaneously with 2 × 10 6 HPD1NR cells. The established tumors were injected i.t. with 1 × 10 9 PFU Ad-TD-nsIL-12 or PBS ( n = 7/group) on day 0, 2, 4, 6, 8, and 10. Control IgG and either mouse anti-hamster CD3 mAb (4F11) a or CD4 mAb b were injected i.p. at doses of 500 μg/injection every fourth day from the day before the viral therapy to the end of the experiment and FACS analysis used to confirm the depletion. Mean tumor volumes and SEM are shown for each group. Statistical analysis was carried out using a one-way ANOVA with post hoc Tukey’s Multiple Comparison Test. * p < 0.05, *** p < 0.001. c – g 2 × 10 6 HPD1NR cells were seeded into the right flank of Syrian hamsters. When tumor volumes reached 300 mm 3 , nine hamsters per group were each injected i.t with PBS, 1 × 10 9 PFU Ad-TD-LUC or Ad-TD- IL-12/nsIL-12 on day 0. On days 3, 7, and 21 tumors were collected and processed for IHC. c Representative images of immunohistochemical staining for CD3 and CD4 at day 7 (×200). d − f Quantitative scores of lymphocyte infiltration within tumors. Lymphocytes were counted in 5 HPFs randomly selected from each tumor section (×200). The scoring was conducted within the tumor and stroma; necrotic areas were avoided. The extent of lymphocyte infiltration was categorized into the following four grades: 1, <25 cells/HPF; 2, 25–49 cells/HPF; 3, 50–75 cells/HPF; 4, >75 cells/HPF. Statistical analysis was carried out using a one-way ANOVA with post hoc Tukey’s Multiple Comparison Test. * p < 0.05, ** p < 0.01, *** p < 0.001. g Spleens and lymph nodes were collected and analyzed by FACS for CD3 and CD4 expression at the time points shown. Mean expression and SEM is plotted ( n = 3/group). Statistical analysis was carried out using a one-way ANOVA with post hoc Tukey’s Multiple Comparison Test. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Splenocytes and lymph node cells (1 × 10 6 ) were prepared and stained with mAb against Syrian hamster CD3 and mAb against mouse CD4-conjugated with FITC (GK1.5, cross-reactive with Syrian hamster, BD), followed by secondary APC-conjugated rabbit anti-mouse antibody for CD3 staining (BD).

Techniques: Injection, Immunohistochemical staining, Staining, Expressing