Journal: PLoS ONE

Article Title: PD-L1 expression in gastroenteropancreatic neuroendocrine neoplasms grade 3

doi: 10.1371/journal.pone.0243900

Figure Lengend Snippet: (a) Red arrow marked immune cell immunoreactive for PD-L1. (b) Red arrow showing same cells immunoreactive for CD3. Scale bar 100 μ m . Inserts, magnification. Scale bars 50 μ m .

Article Snippet: Tumor specimens were stained with a primary monoclonal mouse anti-PD-L1 antibody (PD-L1 IHC clone 22C3 pharm Dx, Agilent, USA) and a CD3 antibody (IR50361-2, FLEX Polyclonal Rabbit Anti-Human CD3, Ready-to-Use (Link), Agilent, USA).

Techniques:

Journal: Nature Communications

Article Title: Topical tacrolimus for the treatment of secondary lymphedema

doi: 10.1038/ncomms14345

Figure Lengend Snippet: ( a ) Representative photographs of mouse tails after surgical excision of superficial/deep collecting lymphatics and treatment with or without topical tacrolimus beginning either 2 weeks (early treatment) or 6 weeks (late treatment) after surgery. Images represent 4 weeks of treatment in the early group and 3 weeks of treatment in the late group. ( b ) Graphical representation of tail volume changes after early (* P =0.021) or late (* P =0.018) treatment with tacrolimus, as compared with controls (red arrows indicate timing of treatment) ( n =8/group). ( c ) Representative cross-sectional histological images (upper panel) of control and early tacrolimus-treated mouse tails harvested 6 weeks after lymphatic ablation. Brackets show soft tissue thickness. Quantification of soft tissue changes (lower panel) after early or late treatment with tacrolimus (* P ≤0.001) ( n =8/group). ( d ) Quantification of whole-blood tacrolimus levels demonstrating immunosuppressive concentrations in systemically treated animals (4 mg kg −1 intraperitoneally daily) and non-immunosuppressive levels in the topically treated group (* P =0.007; n =6/group). ( e ) Representative flow cytometry plots displaying side scatter area (SSA) on the y axis and CD3 + cells (T cells) on the x axis for animals retreated with vehicle control, topical tacrolimus or systemic tacrolimus. ( f ) Quantification of blood T cells for each group. Note significant reduction in T cells only in systemically treated animals (* P =0.012; n =6/group). Experiments were repeated two to three times. All data represent mean±s.d. with P ≤0.05 considered as significant. Two-tailed Student's t -test ( c , d ) and analysis of variance (ANOVA) with post hoc tests ( b , f ).

Article Snippet: Rabbit anti-human/mouse CD3 (A0452; 1:300; Dako, Carpinteria, CA) and Cy3-conjugated mouse anti-αSMA (C6-198; 1:1,000; Sigma-Aldrich).

Techniques: Flow Cytometry, Two Tailed Test

Journal: Oncoimmunology

Article Title: Ectopic CD137 expression by rhabdomyosarcoma provides selection advantages but allows immunotherapeutic targeting

doi: 10.1080/2162402X.2021.1877459

Figure Lengend Snippet: CD137 expression in RMS tissue cores. (a) Percentages of tissue cores with CD137 expression among different types of RMS. Number of (b) CD3 + T cells, (c) CD3 + CD137 + T cells and (d) CD3 − CD137 + cells in different types of RMS. Each symbol represents one patient. Lines represent medians and bars represent interquartile ranges. * p < .05 using Mann Whitney test. ARMS: alveolar rhabdomyosarcoma, ERMS: embryonal rhabdomyosarcoma, PRMS: pleomorphic rhabdomyosarcoma, SC-RMS: Spindle cell-rhabdomyosarcoma

Article Snippet: Primary antibodies used in the Opal staining were mouse anti-human CD137 (clone: BBK2, Thermo Fisher Scientific, Waltham, USA), human anti-human CD137L (clone: P1F3, Genscript), rabbit anti-human CD3 (Dako, Santa Clara, USA), mouse anti-human CD8a (clone: AMC908; Thermo Fisher Scientific), rabbit anti-human ionized calcium-binding adapter molecule-1 (Iba-1) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and mouse anti-human MyoD antibodies (clone: 5.8A; Thermo Fisher Scientific).

Techniques: Expressing, MANN-WHITNEY

Journal: Oncoimmunology

Article Title: Ectopic CD137 expression by rhabdomyosarcoma provides selection advantages but allows immunotherapeutic targeting

doi: 10.1080/2162402X.2021.1877459

Figure Lengend Snippet: Downregulation of CD137 expression on T cells with reduced RMS killing and cytokine secretion. PBMCs isolated from 4 healthy donors were activated with 0.5 ng/ml of anti-CD3 antibody, and cultured with either Rd18-CD137 or parental Rd18 cells for 3 days. Cells were harvested on days 1, 2 and 3 and analyzed by flow cytometry. PBMCs without the RMS cell co-culture provided the baseline. T cells were gated by positive CD3 staining, and RMS cells were gated by negative CD45 staining. Viable RMS cells were defined as CD45 − , Annexin V − , 7-AAD − and the number of viable cells was calculated using counting beads. (a) CD137 and CD69 expression on T cells of one donor is shown. Numbers in the histogram represent percentages of cells with CD137 or CD69 staining. (b) Normalized ratios (Rd18-CD137/Rd18) of percentages for CD137 and CD69 expression. (c) Number of viable RMS cells at day 2 and day 3. (d) Normalized ratios (Rd18-CD137/Rd18) of cytokine levels at day 1, 2 and 3 of co-culture. Each symbol represents one donor. A ratio of 1 (dotted line) indicates no difference between Rd18-CD137 and Rd-18 control. Lines and bars represent means ± standard errors of means. * p < .05 and ** p < .01 using two-sided unpaired t-test

Article Snippet: Primary antibodies used in the Opal staining were mouse anti-human CD137 (clone: BBK2, Thermo Fisher Scientific, Waltham, USA), human anti-human CD137L (clone: P1F3, Genscript), rabbit anti-human CD3 (Dako, Santa Clara, USA), mouse anti-human CD8a (clone: AMC908; Thermo Fisher Scientific), rabbit anti-human ionized calcium-binding adapter molecule-1 (Iba-1) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and mouse anti-human MyoD antibodies (clone: 5.8A; Thermo Fisher Scientific).

Techniques: Expressing, Isolation, Cell Culture, Flow Cytometry, Co-Culture Assay, Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Combination of two fiber-mutant adenovirus vectors, one encoding the chemokine FKN and another encoding cytokine interleukin 12, elicits notably enhanced anti-tumor responses

doi: 10.1007/s00262-008-0499-0

Figure Lengend Snippet: CD3+ T cells infiltration into tumor tissue induced by the intratumoral injection of FKN or/and IL-12. Immunohistochemical analysis was utilized to determine lymphocytes infiltrated into tumors. When the length of the tumor reached about 7–8 mm, intratumoral administrations of indicated Ad were carried out. Tumor-bearing mice were sacrificed 6 days after the intratumoral administration of a AdRGD-Luc, b AdRGD-FKN plus AdRGD-Luc, c AdRGD-IL-12 plus AdRGD-Luc, or d AdRGD-IL-12 plus AdRGD-FKN in total of 4 × 107 PFU all in a ratio of 1:1. The tumor nodules were harvested, embedded in the compound OCT, and stored at −80°C. Frozen thin sections of the nodules were fixed and stained for CD3+ T cells. e The number of immunostained cells was counted under a light microscope with ×400 magnification. For counting the positive cell number that infiltrated into tumor tissue, six fields were randomly selected. Statistical analysis was carried out by Welch’s t test. * <0.05

Article Snippet: The sections were incubated with optimal dilution of the primary antibody, rabbit anti-human CD3 antibody (DakoCytomation, Kyoto, Japan), or normal rabbit IgG (Santa Cruz Biotechnology, USA) for 60 min at room temperature.

Techniques: Injection, Immunohistochemical staining, Staining, Light Microscopy