Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: T cells deficient in the tyrosine phosphatase SHP-1 resist suppression by regulatory T cells 1

doi: 10.4049/jimmunol.1602171

Figure Lengend Snippet: (A) 72-hour proliferation of splenic CellTrace Violet-labeled CD4+CD25- T cells isolated from SHP-1+/+ or SHP-1−/− mice. Proliferation was measured in response to indicated concentrations of anti-CD3 and irradiated CD4+ T cell-depleted splenocytes as APCs. Histograms shown are representative of 3 independent experiments, n=5-7 per genotype. Note that the number of cells on the y axis for histograms is greater for SHP-1−/− T cells than SHP-1+/+ T cells. (B) Percent of CD4+ T cells within each culture initially responding to the indicated stimulation. Data were obtained from the proliferation assays presented in A. Percent of T cell responders was calculated using the precursor frequency algorithm of the FlowJo Proliferation Platform, which takes into account the number of cells in each round of division relative to the input cells, and thereby estimates the fraction of T cells that initially responded to the stimulation. (C) Proliferation assays were set up as described in A, but cells were harvested after 24 hours and assessed for CD25 surface expression. Data represents 3 independent experiments, n=5-9 per genotype. (D) Proliferation index, which corresponds to the average rounds of division of T cells, was obtained from the proliferation assays presented in A using the FlowJo Proliferation Platform. (E) Proliferation assays were set up as described in A. Cells were stained for activated caspase-3 (Casp3) with Fitc-DEVD-FMK for the last hour of culture before harvest and flow cytometric analyses. Data represent percent of Casp3+ cells within CD4+ T cell population, n=3 per genotype. A standard regression ANOVA was performed for B-D, a two-way ANOVA with Sidak’s multiple comparison post-test was used for statistical analysis of E. Error bars indicate ±SEM; * p≤0.05, **p≤0.01, ***p≤0.001.

Article Snippet: Analysis of Proliferation Assay CellTrace Violet dilution was assessed by flow cytometry, and subsequently analyzed using FlowJo v 9.9 Software Proliferation Wizard Platform (FlowJo, LLC.)

Techniques: Labeling, Isolation, Irradiation, Expressing, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: T cells deficient in the tyrosine phosphatase SHP-1 resist suppression by regulatory T cells 1

doi: 10.4049/jimmunol.1602171

Figure Lengend Snippet: (A) Splenic CD4+CD25- T (Tcon) cells were isolated from SHP-1+/+ or SHP-1−/− mice, labeled with CellTrace Violet and cultured either alone or with wild type Tregs at the indicated ratios in the presence of 150ng/mL anti-CD3 and irradiated CD4+ T cell-depleted splenocytes as APCs, and proliferation was measured after 4 days. Histograms shown are representative of 5 independent experiments, n=8–10 mice per genotype. Bold numbers indicate most significant differences observed. (B) Suppression was calculated by normalizing each data point to the corresponding baseline proliferation (no Tregs, maximal response = 100% proliferation), which was then subtracted from 100 % proliferation. Note that as described in Fig. 1, proliferation was computed using FlowJo Proliferation Platform, which takes into account the number of cells in each round of division relative to the input cells, and thereby estimates the fraction of T cells that initially responded to the stimulation. A three-way ANOVA was performed. (C) Naïve CD4+CD44lo T cells were purified from spleens of SHP-1+/+ or SHP-1−/− mice and suppression assays were performed as described in A in the presence of 30 ng/mL anti-CD3 and CD4+ T cell-depleted splenocytes as APCs, n=3 mice per genotype. (D) Suppression was calculated as in B. Student’s t tests were performed for each Treg:T cell ratio. Error bars indicate ±SEM; * p≤0.05, **p≤0.01, ***p≤0.001.

Article Snippet: Analysis of Proliferation Assay CellTrace Violet dilution was assessed by flow cytometry, and subsequently analyzed using FlowJo v 9.9 Software Proliferation Wizard Platform (FlowJo, LLC.)

Techniques: Isolation, Labeling, Cell Culture, Irradiation, Purification

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: T cells deficient in the tyrosine phosphatase SHP-1 resist suppression by regulatory T cells 1

doi: 10.4049/jimmunol.1602171

Figure Lengend Snippet: (A) Splenic CD8+ T cells were isolated from SHP-1+/+ or SHP-1−/− mice, labeled with CellTrace Violet and cultured either alone or with wild type Tregs at the indicated ratios in the presence of 10ng/mL anti-CD3 and CD4+ T cell-depleted splenocytes as APCs, and proliferation was measured after 3 days. Data are representative of 2 independent experiments; n=4 mice per genotype. (B) The percent responding cells was obtained using FlowJo Proliferation Platform as described in Figs. 1 and ​and2.2. Suppression was calculated by normalizing each data point to the corresponding baseline proliferation (no Tregs, maximal response = 100% proliferation), which was then subtracted from 100 % proliferation. A three-way ANOVA was performed. (C) Naïve CD8 T cells (CD8+CD44lo) were isolated, labeled with CellTrace Violet, and cultured with Tregs as described in A. n=3 mice per genotype. (D) Percent suppression was obtained as in B. Student’s t tests were performed for each Treg:T cell ratio. Error bars indicate ±SEM. * p≤0.05, **p≤0.01.

Article Snippet: Analysis of Proliferation Assay CellTrace Violet dilution was assessed by flow cytometry, and subsequently analyzed using FlowJo v 9.9 Software Proliferation Wizard Platform (FlowJo, LLC.)

Techniques: Isolation, Labeling, Cell Culture

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: T cells deficient in the tyrosine phosphatase SHP-1 resist suppression by regulatory T cells 1

doi: 10.4049/jimmunol.1602171

Figure Lengend Snippet: (A) Schematic representation of experimental setup. Splenic CD4+CD25- T cells (Tcon cells) were isolated from SHP-1+/+ or SHP-1−/− mice and labeled with CellTrace dyes. Differently-labeled Tcon cells of indicated genotypes were co-cultured at 1:1 ratio in the presence of 30ng/mL anti-CD3 and irradiated CD4+ T cell-depleted splenocytes as APCs. (B) After 72 hours, the proliferation of the CellTrace Violet-labeled cells was measured and assessed using the FlowJo Proliferation platform as in Fig. 1. Graph shows percent responding cells of indicated genotype, compiled from two independent experiments; n= 6 mice per genotype. (C) Using the same setup as in B, with the addition of wild type Tregs at a ratio of 1:4 of Treg:total Tcon cells. After 96 hours, proliferation of CellTrace Violet-labeled cells was measured and analyzed as in B. Graph shows percent of suppression (calculated as in Figs. 2 and ​and3).3). A one-way ANOVA was performed on data in B and C. Error bars indicate ±SEM; * p≤0.05.

Article Snippet: Analysis of Proliferation Assay CellTrace Violet dilution was assessed by flow cytometry, and subsequently analyzed using FlowJo v 9.9 Software Proliferation Wizard Platform (FlowJo, LLC.)

Techniques: Isolation, Labeling, Cell Culture, Irradiation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: T cells deficient in the tyrosine phosphatase SHP-1 resist suppression by regulatory T cells 1

doi: 10.4049/jimmunol.1602171

Figure Lengend Snippet: (A) Schematic representation of experimental setup. Splenic CD4+CD25- Tcon cells were isolated from wild type CD45.1 mice or SHP-1+/+ or SHP-1−/− CD45.2 mice and labeled with CellTrace Violet. Wild type Tregs (CD4+CD25+) were isolated from spleens of SHP-1+/+ mice. 3×106 total Tcon cells were injected i.v. via the tail vein into Rag1−/− recipient mice, at a 1:1 ratio of either CD45.2 SHP-1+/+:CD45.1 wild type Tcon cells or CD45.2 SHP-1−/−:CD45.1 wild type Tcon cells. Half the recipients received Tcon cells only, and the other half received Tcon cells along with 7.5×105 SHP-1+/+ Tregs (1:4 Treg:Tcon ratio). After 10 days, spleens of recipient mice were harvested and stained for analysis by flow cytometry. (B) (Left) Representative flow plots of CD4+CD25- input Tcon cells. (Top) Input for conditions i and ii: CD45.2 SHP-1+/+ with CD45.1 wild type Tcon cells; (Bottom) input for conditions iii and iv: CD45.2 SHP-1−/− with CD45.1 wild type Tcon cells. (Right) Plots show percentages of splenic CD45.1+ and CD45.2+ CD4+Foxp3- T cells recovered 10 days post-injection; experimental conditions (with or without Tregs) as indicated. (C) Percent suppression was computed by subtracting the percent relative expansion for each indicated genotype from 100%. Percent relative expansion was calculated by dividing the absolute number of CD4+Foxp3- T cells recovered in the presence of co-injected Tregs over the absolute number of CD4+Foxp3- T cells recovered in the absence of Tregs (maximal expansion), multiplied by 100. n=3-4 recipient mice per donor condition. Error bars indicate ±SEM; * p≤0.05.

Article Snippet: Analysis of Proliferation Assay CellTrace Violet dilution was assessed by flow cytometry, and subsequently analyzed using FlowJo v 9.9 Software Proliferation Wizard Platform (FlowJo, LLC.)

Techniques: Isolation, Labeling, Injection, Staining, Flow Cytometry