Journal: bioRxiv

Article Title: The adenosine 2a receptor and TIM3 directly inhibit killing of tumor cells by cytotoxic T lymphocytes through interference with cytoskeletal polarization

doi: 10.1101/2021.05.20.444944

Figure Lengend Snippet: Mouse numbers across the different parts of the experiments are detailed in Fig. S2A. A RencaHA tumor-bearing BALB/c were injected i.v. with two doses of 5 × 106 Thy1.1+ CL4 CTL (ATT) plus either vehicle and isotype control, Aa2R-Antagonist (ZM 241385) alone or A2aR-antagonist + anti-TIM3 mAb as shown. The percentage of Thy1.1+ CL4 TIL between days 25 and 48 is given as representative data from N=25 mice over 3 independent experiments. Mice were either immunized with HA peptide 5 days prior to TIL harvest (N=5) to induce expansion of Thy1.1+ CL4 T cells or immunized with empty vehicle control (N=19); one representative graph is shown for each group. B-E Tumor growth curves from individual mice treated in , which had experienced complete and durable tumor remission. B untreated and C rechallenged with tumor cells at day 40; D depleted of Thy1.1+ T cells using anti-Thy1.1 depleting mAb at 28 days; E depleted of all CD8+ T cells using anti-CD8β depleting mAb at 28 days with depletion efficiency shown in Fig. S2B.

Article Snippet: Antibodies used are described in the order: antigen, fluorescent label, clone, supplier, dilution: For flow cytometry: FcBlock no azide (for blockade of Fc receptors) 2.4G2 BD Biosciences 1:50 CD8α FITC 53-6.7 BD Bioscience 1:100 CD8β PeCy7 YTS156.7.7 Biolegend 1:200 CD4 AF700 CKI1.5 Biolegend 1:100 CD39 PerCP-Cy5.5 24DMSI eBioscience 1:100 CD73 BV605 T111.8 Biolegend 1:100 TIM3 PE B8.2C1.2 Biolegend 1:100 TIM3 BV605 RMT3-23 Biolegend 1:100 TIGIT APC 1G9 Biolegend 1:100 LAG3 PeCy7 C9B7W eBioscience 1:200 PD1 BV785 29F.1A12 Biolegend 1:200 TCRβ AF647 H57-597 Biolegend 1:200 Thy1.1 FITC OX7 BD Bioscience 1:100 Thy1.1 PerCP-Cy5.5 OX7 Biolegend 1:100 CEACAM1 APC CC1 Biolegend 1:100 For blocking and T cell priming TIM3 no azide (for in vitro/in vivo blockade) RMT3-23 BioXcell In Vivo mAb in vivo: 100μg/mouse in vitro: 10μg/ml Isotype control for anti-TIM3 Rat IgG2a 2A3 no azide (for in vivo/in vitro blockade) BioXcell In Vivo mAb in vivo: 100μg/mouse in vitro: 10μg/ml CD8β no azide (for in vivo depletion) 53-5.8 BioXcell InVivoMAb 100μg/mouse Thy1.1 no azide (for in vivo depletion) 19E12 BioXcell InVivoMAb 250μg/mouse CD3ɛ no azide (for in vitro priming) 145-2C11 BioXcell InVivoMAb 10μg/ml CD28 no azide (for in vitro priming) 37.51 BioXcell InVivoMAb 1μg/ml For immunohistochemistry: FcBlock no azide (for blockade of Fc receptors) 2.4G2 BD Biosciences 1:50 CD8α no azide 53-6.7 Biolegend 1:500 Rabbit H+L AF488 Life Technologies 1:1000 Rabbit H+L AF405 Life Technologies 1:1000 Rat IgG2a,α Biolegend 1:500 Rat IgG H+L AF594 ThermoFisher 1:2000 FOXP3 no azide FJK-16S ThermoFisher 1:100 FOXP3 APC FJK-16S ThermoFisher 1:40 Thy1.1 FITC OX7 BD Bioscience 1:100 Isotype control for Thy1.1 Mouse IgG1,α FITC BD Bioscience 1:100 CD19 (dump) BV510 6D5 Biolegend 1:100 TCRβ SB645 H57-597 ThermoFisher 1:200 CD4 PE-Cy5.5 RM4-5 ThermoFisher 1:3000 CD8β PE-Cy5 H35-17.2 ThermoFisher 1:3000 CD25 VioBright-FITC 7D4 Miltenyi 1:200 CD73 BV605 TY/11.8 Biolegend 1:100 CD39 PerCP-eFluor710 24DMS1 ThermoFisher 1:100

Techniques: Injection

Journal: bioRxiv

Article Title: The adenosine 2a receptor and TIM3 directly inhibit killing of tumor cells by cytotoxic T lymphocytes through interference with cytoskeletal polarization

doi: 10.1101/2021.05.20.444944

Figure Lengend Snippet: A-E Ex vivo cytoskeletal polarization of CL4 TIL from RencaHA tumor-bearing BALB/c mice upon ATT of CL4 T cells and treatment with combinations of A2aR-Antagonist and anti-TIM3 mAb (N=5 control, N=3 both treatments, N=2 AR-Antagonist alone) in comparison to in vitro CL4 CTL is given. Mouse numbers across the different parts of the experiments are detailed in Fig. S2A. A Representative images of an off-interface lamella with the immune synapse in red and an off-interface lamella in blue. Scale bar=8μm. B Time until formation of the first off-interface lamella is given. Kaplan-Meier survival analysis (Log Rank) N>30 cell couples per condition over 4 experiments. C Translocation is shown as one CTL (red circle) moves >1 interface diameter from the initial location of the immune synapse (green) between early (left panel) and late (right panel) timepoints. Scale bar=8μm. D The frequency of cell couples with translocation is given. Pairwise proportion’s z-test. N>30 cell couples per condition over 4 experiments. E Growth curves of the tumors used for analyses A-D are shown. F RencaHA tumor-bearing BALB/c mice were treated with A2aR antagonist and anti-TIM3 mAb from day 12. ATT of CL4 T cells was given on day 12. Killing of KdHA-pulsed Renca mCherry tumor cells by day 16 ex vivo CL4 TIL is given at an E:T ratio of 3:2. Each point=1 tumor, N=2 experiments. Size-matched tumors analyzed on the same day are paired for comparison using a t-test. G Growth of the tumors used for killing analysis in F are shown as two separate experiments. H, I Half of size-matched tumors from mice in E and G were stained. H A representative image with CD8 staining in red and Thy1.1 staining in green. scale bar=50μm. I The numbers of (left panel) endogenous Thy1.2+CD8+ TIL and adoptively transferred Thy1.1+ Clone4 TIL or (right panel) total CD8+ TIL and FOXP3+ regulatory T cells in 10 peripheral and 10 central tumor areas are given. N=2 control tumors and 3 treated tumors analyzed over two experiments. Size matched tumors fixed on the same day are paired for analysis using t-test.

Article Snippet: Antibodies used are described in the order: antigen, fluorescent label, clone, supplier, dilution: For flow cytometry: FcBlock no azide (for blockade of Fc receptors) 2.4G2 BD Biosciences 1:50 CD8α FITC 53-6.7 BD Bioscience 1:100 CD8β PeCy7 YTS156.7.7 Biolegend 1:200 CD4 AF700 CKI1.5 Biolegend 1:100 CD39 PerCP-Cy5.5 24DMSI eBioscience 1:100 CD73 BV605 T111.8 Biolegend 1:100 TIM3 PE B8.2C1.2 Biolegend 1:100 TIM3 BV605 RMT3-23 Biolegend 1:100 TIGIT APC 1G9 Biolegend 1:100 LAG3 PeCy7 C9B7W eBioscience 1:200 PD1 BV785 29F.1A12 Biolegend 1:200 TCRβ AF647 H57-597 Biolegend 1:200 Thy1.1 FITC OX7 BD Bioscience 1:100 Thy1.1 PerCP-Cy5.5 OX7 Biolegend 1:100 CEACAM1 APC CC1 Biolegend 1:100 For blocking and T cell priming TIM3 no azide (for in vitro/in vivo blockade) RMT3-23 BioXcell In Vivo mAb in vivo: 100μg/mouse in vitro: 10μg/ml Isotype control for anti-TIM3 Rat IgG2a 2A3 no azide (for in vivo/in vitro blockade) BioXcell In Vivo mAb in vivo: 100μg/mouse in vitro: 10μg/ml CD8β no azide (for in vivo depletion) 53-5.8 BioXcell InVivoMAb 100μg/mouse Thy1.1 no azide (for in vivo depletion) 19E12 BioXcell InVivoMAb 250μg/mouse CD3ɛ no azide (for in vitro priming) 145-2C11 BioXcell InVivoMAb 10μg/ml CD28 no azide (for in vitro priming) 37.51 BioXcell InVivoMAb 1μg/ml For immunohistochemistry: FcBlock no azide (for blockade of Fc receptors) 2.4G2 BD Biosciences 1:50 CD8α no azide 53-6.7 Biolegend 1:500 Rabbit H+L AF488 Life Technologies 1:1000 Rabbit H+L AF405 Life Technologies 1:1000 Rat IgG2a,α Biolegend 1:500 Rat IgG H+L AF594 ThermoFisher 1:2000 FOXP3 no azide FJK-16S ThermoFisher 1:100 FOXP3 APC FJK-16S ThermoFisher 1:40 Thy1.1 FITC OX7 BD Bioscience 1:100 Isotype control for Thy1.1 Mouse IgG1,α FITC BD Bioscience 1:100 CD19 (dump) BV510 6D5 Biolegend 1:100 TCRβ SB645 H57-597 ThermoFisher 1:200 CD4 PE-Cy5.5 RM4-5 ThermoFisher 1:3000 CD8β PE-Cy5 H35-17.2 ThermoFisher 1:3000 CD25 VioBright-FITC 7D4 Miltenyi 1:200 CD73 BV605 TY/11.8 Biolegend 1:100 CD39 PerCP-eFluor710 24DMS1 ThermoFisher 1:100

Techniques: Ex Vivo, In Vitro, Translocation Assay, Staining

Journal: Stem Cells International

Article Title: Characterization of Progenitor Cells during Canine Retinal Development

doi: 10.1155/2012/675805

Figure Lengend Snippet: Antibodies and dilutions.

Article Snippet: Mouse monoclonal to Thy1.1 , Abcam, MA, USA (ab65193) , 1 : 200.

Techniques: