Journal: eLife

Article Title: T cell self-reactivity during thymic development dictates the timing of positive selection

doi: 10.7554/eLife.65435

Figure Lengend Snippet: ( a ) Expression of CD5 on CXCR4−CCR7 + CD4 CD8+TG6 (96 hr), F5 (72 hr and 96 hr), and OT-1 (72 hr and 96 hr) donor cells developed in thymic slices. Values for each experiment are normalized to the average CD5 expression of the CXCR4−CCR7 + CD4 CD8+polyclonal slice resident (SR) population at the same time points. ( b–d ) Preselection TG6 (white bars, triangle), F5 (gray bars, square), or OT-1 (dark grey bars, circle) thymocytes were overlaid onto selecting or nonselecting thymic slices, harvested at 2, 24, 48, 72, or 96 hr post-thymocyte overlay, and analyzed using flow cytometry. Left three graphs show individual values for each transgenic. Horizontal lines in ( a ) and ( b ) indicate the average value for nonselecting slices. Right line graph shows the average for all three transgenics overlaid. ( b ) Percent of CD69+ cells within the CD4+CD8+ and CD4−CD8+populations. ( c ) Percentage of CXCR4−CCR7 + cells within CD4+CD8+ and CD4−CD8+populations. ( d ) Percentage of CXCR4−CCR7 + CD4 CD8+ cells out of the donor population. Values for each experiment are normalized to the average at the time point with the maximum CD8SP development (72 hr or 96 hr). For ( a ), data are presented as average ± SD and analyzed using an ordinary one-way ANOVA with Tukey’s multiple comparisons (****p<0.000). For ( b–d ), data are presented as average ± SD and analyzed using an ordinary one-way ANOVA with Dunnett’s multiple comparisons (**p<0.01, ***p<0.001, and ****p<0.0001) comparing to 2 hr time point. All data are compiled from three or more experiments. Preselection thymocyte populations were obtained from TG6tg Rag2 −/− H2 b mice, irradiated β2M −/− mice reconstituted with F5tg Rag1 −/− bone marrow, and OT-1tg Rag2 −/− β2M −/− mice. See also and .

Article Snippet: Antibody , Rat Monoclonal anti-Mouse CD8α (Clone 53–6.7) PE-Cy7 , Tonbo Biosciences , Cat. No. 60–0081 U100 RRID: AB_2621832 , (1:200).

Techniques: Expressing, Flow Cytometry, Transgenic Assay, Irradiation

Journal: eLife

Article Title: T cell self-reactivity during thymic development dictates the timing of positive selection

doi: 10.7554/eLife.65435

Figure Lengend Snippet: ( a ) Representative flow plots illustrating the gating strategy for thymic slice experiments. ( b ) Representative CD4 vs CD8 flow plots of TG6, F5, and OT-1 live donor thymocytes (bolded red gate in a ). ( c ) CD4 vs CD8 representative flow plots for TG6, F5, and OT-1 live donor CXCR4-CCR7 + thymocytes (bolded blue gate in a ). Bolded numbers within plots indicate the percentage of cells within a given gate.

Article Snippet: Antibody , Rat Monoclonal anti-Mouse CD8α (Clone 53–6.7) PE-Cy7 , Tonbo Biosciences , Cat. No. 60–0081 U100 RRID: AB_2621832 , (1:200).

Techniques:

Journal: eLife

Article Title: T cell self-reactivity during thymic development dictates the timing of positive selection

doi: 10.7554/eLife.65435

Figure Lengend Snippet: Thymuses were harvested from TG6 (triangle, white bar), F5 (square, gray bar), or OT-1 (circle, black bar) transgenic neonatal mice at 7, 14, and 21 days post-birth and from adult mice (6–9 weeks old), and analyzed using flow cytometry. ( a ) Percentage of CD4−CD8+ cells. ( b ) Total number of CD4−CD8+ thymocytes at the indicated time point post-birth, relative to adult, for each transgenic. ( c ) Percentage of CXCR4−CCR7+ cells within CD4+CD8+ and CD4−CD8+ populations. Data are presented as average ± SD and analyzed using an ordinary one-way ANOVA, Dunnett’s multiple comparisons (*p<0.05, **p<0.01, and ****p<0.0001) comparing to adult. All data are compiled from three or more experiments.

Article Snippet: Antibody , Rat Monoclonal anti-Mouse CD8α (Clone 53–6.7) PE-Cy7 , Tonbo Biosciences , Cat. No. 60–0081 U100 RRID: AB_2621832 , (1:200).

Techniques: Transgenic Assay, Flow Cytometry

Journal: eLife

Article Title: T cell self-reactivity during thymic development dictates the timing of positive selection

doi: 10.7554/eLife.65435

Figure Lengend Snippet: Total number of CD4−CD8+ splenocytes relative to adult for each transgenic. Data compiled from three or more experiments.

Article Snippet: Antibody , Rat Monoclonal anti-Mouse CD8α (Clone 53–6.7) PE-Cy7 , Tonbo Biosciences , Cat. No. 60–0081 U100 RRID: AB_2621832 , (1:200).

Techniques: Transgenic Assay

Journal: eLife

Article Title: T cell self-reactivity during thymic development dictates the timing of positive selection

doi: 10.7554/eLife.65435

Figure Lengend Snippet: TG6, F5, or OT-1 ( a–c ) or B6 ( d–f ) mice were injected with two doses of 1 mg of EdU intraperitoneally (i.p.) at 0 and 4 hr post-injection (p.i.). ( a ) TCRtg experimental schematic. ( b ) EdU incorporation into TCRtg thymuses. ( c ) (Left three graphs) Percent CD8SP and DP thymocytes out of gated EdU+ cells at various days p.i. in TG6, F5, and OT-1 tg thymuses. (Right) The percent of EdU+CD8 SP relative to max within each experiment. Data are presented as the average within each transgenic and analyzed using an ordinary one-way ANOVA at each time point (*p<0.05, **p<0.01). Tukey’s multiple comparisons showed significant differences between OT-1 and TG6 at day 4 (**), day 5 (*), and day 6 (*); OT-1 and F5 at day 4 (*); and a difference between F5 vs TG6 at Day 6 (p=0.069). ( d ) B6 experimental schematic. ( e ) Representative gating strategy and histograms showing B6 day 10 p.i. CD5 expression on EdU+ and EdU− mature SP cells. ( f ) CD5 expression of EdU+TCRβ+CD8 SP and EdU+CD4 SP thymocytes at 4, 7, and 10 days p.i. CD5 expression shown relative to total TCRb+CD8 SP or CD4SP CD5 expression. In ( f ), data are presented as average ± SD and analyzed using an ordinary one-way ANOVA, Tukey’s multiple comparisons (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). All data are compiled from three or more experiments.

Article Snippet: Antibody , Rat Monoclonal anti-Mouse CD8α (Clone 53–6.7) PE-Cy7 , Tonbo Biosciences , Cat. No. 60–0081 U100 RRID: AB_2621832 , (1:200).

Techniques: Injection, Transgenic Assay, Expressing

Journal: eLife

Article Title: T cell self-reactivity during thymic development dictates the timing of positive selection

doi: 10.7554/eLife.65435

Figure Lengend Snippet: CD5 gMFI of OT-1, F5, and TG6 EdU+CD8 SP thymocytes at the indicated time point post-EdU injection. Data is normalized to day 4 for OT-1 and F5, and to day 5 for TG6. CD5 gMFI is not significantly different within each transgenic regardless of the day post-injection. Data are presented as average ± SD and analyzed using an ordinary one-way ANOVA, Tukey’s multiple comparisons. All data are compiled from three or more experiments.

Article Snippet: Antibody , Rat Monoclonal anti-Mouse CD8α (Clone 53–6.7) PE-Cy7 , Tonbo Biosciences , Cat. No. 60–0081 U100 RRID: AB_2621832 , (1:200).

Techniques: Injection, Transgenic Assay

Journal: eLife

Article Title: T cell self-reactivity during thymic development dictates the timing of positive selection

doi: 10.7554/eLife.65435

Figure Lengend Snippet:

Article Snippet: Antibody , Rat Monoclonal anti-Mouse CD8α (Clone 53–6.7) PE-Cy7 , Tonbo Biosciences , Cat. No. 60–0081 U100 RRID: AB_2621832 , (1:200).

Techniques: Generated, Staining, Flow Cytometry, Software

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The role of Ly49E receptor expression on murine intraepithelial lymphocytes in intestinal cancer development and progression

doi: 10.1007/s00262-016-1894-6

Figure Lengend Snippet: Tumor-infiltrating T cells and tumor uPA expression in AOM-treated Ly49E WT versus Ly49E KO mice. a Hematoxylin/eosin-(H&E)- (upper), and CD3-(lower) stained paraffin tumor sections from AOM-treated Ly49E WT and Ly49E KO mice. Scale bar 250 µm, ×100 magnification. A graph showing CD3 mean gray value/mm2 according to tumor surface area (mm2) is shown for AOM-treated Ly49E WT and Ly49E KO mice (n = 6). b Colon tumor-infiltrating IEL subpopulation frequencies in AOM-treated Ly49E WT and Ly49E KO mice 14–22 weeks following the start of treatment, and colon IEL subpopulation frequencies from untreated Ly49E WT and Ly49E KO mice (mean ± SEM; n = 5 for AOM-treated mice; n = 3 for untreated mice). The percentage of TCRαβ and TCRγδ IEL is shown as a fraction of the total numbers of T cells. TCRαβ CD4, TCRαβ CD8αβ and TCRαβ CD8αα IEL subpopulation frequencies are shown as a percentage of the total TCRαβ IELs. TCRγδ DN and TCRγδ CD8αα IEL subpopulation frequencies are shown as a percentage of the total TCRγδ IEL. c Dot plots are shown for CD4/CD8β versus CD8α expression in colon tumor-infiltrating IEL in AOM-treated Ly49E WT and Ly49E KO mice, and colon IEL from untreated Ly49E WT and Ly49E KO mice. Numbers indicate the percentage of cells in each quadrant. Dot plots are representative for n = 5 AOM-treated mice and n = 3 untreated mice. d Tumor uPA expression in tumors of varying size from AOM-treated Ly49E WT and Ly49E KO mice at 14–22 weeks following the start of treatment. n.s. not significant. Data were analyzed using the nonparametric two-tailed Mann–Whitney U-test or the Kruskall–Wallis test. A p value ≤0.05, (*), a p value ≤0.01 (**) and p value ≤0.001 (***), were considered statistically significant

Article Snippet: Anti-CD8α (PE/Cy7-conjugated, clone 53-6.7) from eBioscience (San Diego, CA, USA).

Techniques: Expressing, Staining, Two Tailed Test, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Murine precursors to type 1 conventional dendritic cells induce tumor cytotoxicity and exhibit activated PD-1/PD-L1 pathway

doi: 10.1371/journal.pone.0273075

Figure Lengend Snippet: Antibodies used for flow cytometry.

Article Snippet: Pooled DCs were washed in flow buffer (PBS with 0.5% FBS), incubated with anti-mouse Fc Block (Thermo Fisher Scientific), and then stained with CD8α PE-Cy7 (Thermo Fisher; clone: 53–6.7), and CD24 Pacific Blue (Biolegend; clone: M1/69).

Techniques: Cytometry

Journal: PLoS ONE

Article Title: Murine precursors to type 1 conventional dendritic cells induce tumor cytotoxicity and exhibit activated PD-1/PD-L1 pathway

doi: 10.1371/journal.pone.0273075

Figure Lengend Snippet: Splenic pan DCs were isolated from naïve BALB/c mice and analyzed by flow cytometry to characterize pre-cDC1s and CD8α+ cDC1s. (A) Representative flow plot demonstrating gating strategy. Gated on CD11c+B220- (conventional DCs); pre-cDC1s are gated as CD24 high CD8α- and CD8α+ cDC1s are gated as CD8α+. (B) Mean absolute cell number of respective DC subsets shown with SEM. (C-F) Mean percent and MFI with representative histograms shown with SEM for expression of (C) PD-L1, (D) PIR-B, (E) CD70, and (F) ICOSL on CD8α+ cDC1s (black) and pre-cDC1s (cyan). Data is representative of two experiments. (n = 5 per group). Paired t tests were used to determine significance between DC subsets. **P<0.01, ***P<0.001.

Article Snippet: Pooled DCs were washed in flow buffer (PBS with 0.5% FBS), incubated with anti-mouse Fc Block (Thermo Fisher Scientific), and then stained with CD8α PE-Cy7 (Thermo Fisher; clone: 53–6.7), and CD24 Pacific Blue (Biolegend; clone: M1/69).

Techniques: Isolation, Flow Cytometry, Expressing

Journal: PLoS ONE

Article Title: Murine precursors to type 1 conventional dendritic cells induce tumor cytotoxicity and exhibit activated PD-1/PD-L1 pathway

doi: 10.1371/journal.pone.0273075

Figure Lengend Snippet: BALB/c mice were dosed with 200 μg of 2.43 mAb antibody intraperitoneally on day -3 and day -1. On day 0, splenic DCs were isolated and analyzed by flow cytometry. Data is representative of three independent experiments. (n = 4 per group) (A) Representative flow plots depicting relative cDC1 proportions in naïve DCs (left) and 2.43 mAb DCs (right). Gated on CD11c+B220- (conventional DCs). (B) Mean percent of CD8α+ cDC1s (black) and pre-cDC1s (cyan) in untreated and 2.43 mAb-treated DCs shown with SEM. Two-way ANOVA and Dunnett’s multiple comparisons used to determine significance. **P<0.01, ****P<0.0001. (C) Mean absolute cell number of pre-cDC1s (CD24 high CD8α-) in untreated (black) and 2.43 mAb-treated (magenta) groups shown with SEM. Unpaired t test used to determine significance. *P<0.05. (D) Schematic depicting experimental design for proposed allogeneic mixed leukocyte reaction (MLR) using 2.43 mAb treatment.

Article Snippet: Pooled DCs were washed in flow buffer (PBS with 0.5% FBS), incubated with anti-mouse Fc Block (Thermo Fisher Scientific), and then stained with CD8α PE-Cy7 (Thermo Fisher; clone: 53–6.7), and CD24 Pacific Blue (Biolegend; clone: M1/69).

Techniques: Isolation, Flow Cytometry

Journal: PLoS ONE

Article Title: Murine precursors to type 1 conventional dendritic cells induce tumor cytotoxicity and exhibit activated PD-1/PD-L1 pathway

doi: 10.1371/journal.pone.0273075

Figure Lengend Snippet: BALB/c mice were dosed with 200 μg of 2.43 mAb antibody intraperitoneally on day -3 and day -1. On day 0, splenic DCs were isolated and plated in an allogeneic MLR. DC composition was determined by flow cytometry on day 2 of co-culture. Data is representative of two independent experiments. (n = 4 per group) (A) Representative flow plots depicting percent of H2K d +CD11c+ DCs in naïve DCs (left) and 2.43 mAb DCs (right) on day 2 of co-culture. (B) Mean percent of H2K d +CD11c+ DCs for naïve (black) or 2.43 mAb DCs (magenta) shown with SEM. Unpaired t test was used to determine significance between groups. **P<0.01. (C) Representative flow plots depicting percent CD8α+ cDC1s and pre-cDC1s in naïve (left) and 2.43 mAb DCs (right). (D) Mean percent CD8α+ cDC1s (black bars) and pre-cDC1s (teal bars) in naïve and 2.43 mAb DCs shown with SEM. 2way ANOVA and Šidák’s multiple comparison used to determine statistical significance. **P<0.01.

Article Snippet: Pooled DCs were washed in flow buffer (PBS with 0.5% FBS), incubated with anti-mouse Fc Block (Thermo Fisher Scientific), and then stained with CD8α PE-Cy7 (Thermo Fisher; clone: 53–6.7), and CD24 Pacific Blue (Biolegend; clone: M1/69).

Techniques: Isolation, Flow Cytometry, Co-Culture Assay

Journal: PLoS ONE

Article Title: Murine precursors to type 1 conventional dendritic cells induce tumor cytotoxicity and exhibit activated PD-1/PD-L1 pathway

doi: 10.1371/journal.pone.0273075

Figure Lengend Snippet: Splenic DCs were isolated from naïve BALB/c mice and pre-cDC1s and CD8α+ cDC1s were cell sorted for RNA sequencing. (A) Enhanced volcano plot identifying genes that are up or down-regulated in pre-cDC1s compared to CD8α+ cDC1s. (B-C) Pathway results using Qiagen’s IPA software. P = 0.05 corresponds to a 1.3 -log p-value, and anything above this value is considered statistically significant. (B) Pathways predicted to be significantly inhibited (z-score < -2) in pre-cDC1s compared to CD8α+ cDC1s. (C) Pathways predicted to be significantly activated (z-score < 2) in pre-cDC1s compared to CD8α+ cDC1s. (D) Heat map showing relative gene expression levels for the PD-1/PD-L1 cancer immunotherapy pathway. Full documentation, methodology, and raw data for this data set is available online at: https://doi.org/10.25422/azu.data.14241902 .

Article Snippet: Pooled DCs were washed in flow buffer (PBS with 0.5% FBS), incubated with anti-mouse Fc Block (Thermo Fisher Scientific), and then stained with CD8α PE-Cy7 (Thermo Fisher; clone: 53–6.7), and CD24 Pacific Blue (Biolegend; clone: M1/69).

Techniques: Isolation, RNA Sequencing Assay, Software, Expressing