Structured Review

Becton Dickinson anti cd4
Autologous suppression by human nTreg cells. (a and b) Ability of nTreg cells freshly isolated from WAS patients and HDs to suppress autologous <t>CD4</t> + CD25 − effector T cells. Effector T cells were cultured in the presence of nTreg cells at the indicated ratio and stimulated with CD3-depleted irradiated allogeneic APCs and anti-CD3 mAbs for 72 h (HD1 and WAS4), or with anti-CD3 mAbs plus anti-CD28 mAb–coated beads for 120 h (HD5 and WAS21). Proliferation was measured by [ 3 H]thymidine incorporation (a), and IFN-γ secretion was measured by cytometric bead array (b). Filled bars, activated effector T cells (abbreviated as E); gray bars, activated effector T cells in the presence of nTreg cells isolated from HDs; dashed bars, activated effector T cells in the presence of nTreg cells isolated from WAS patients; dotted bars, activated nTreg cells alone. Percentages of suppression are indicated. (c and d) Cumulative graph of autologous suppression of proliferation (c) and IFN-γ production (d) at the indicated effector T cell/nTreg cell ratio. Dots represent percentage of suppression of each HD or WAS patient, and bars depict the respective median value.
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1) Product Images from "WASP regulates suppressor activity of human and murine CD4+CD25+FOXP3+ natural regulatory T cells"

Article Title: WASP regulates suppressor activity of human and murine CD4+CD25+FOXP3+ natural regulatory T cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20061334

Autologous suppression by human nTreg cells. (a and b) Ability of nTreg cells freshly isolated from WAS patients and HDs to suppress autologous CD4 + CD25 − effector T cells. Effector T cells were cultured in the presence of nTreg cells at the indicated ratio and stimulated with CD3-depleted irradiated allogeneic APCs and anti-CD3 mAbs for 72 h (HD1 and WAS4), or with anti-CD3 mAbs plus anti-CD28 mAb–coated beads for 120 h (HD5 and WAS21). Proliferation was measured by [ 3 H]thymidine incorporation (a), and IFN-γ secretion was measured by cytometric bead array (b). Filled bars, activated effector T cells (abbreviated as E); gray bars, activated effector T cells in the presence of nTreg cells isolated from HDs; dashed bars, activated effector T cells in the presence of nTreg cells isolated from WAS patients; dotted bars, activated nTreg cells alone. Percentages of suppression are indicated. (c and d) Cumulative graph of autologous suppression of proliferation (c) and IFN-γ production (d) at the indicated effector T cell/nTreg cell ratio. Dots represent percentage of suppression of each HD or WAS patient, and bars depict the respective median value.
Figure Legend Snippet: Autologous suppression by human nTreg cells. (a and b) Ability of nTreg cells freshly isolated from WAS patients and HDs to suppress autologous CD4 + CD25 − effector T cells. Effector T cells were cultured in the presence of nTreg cells at the indicated ratio and stimulated with CD3-depleted irradiated allogeneic APCs and anti-CD3 mAbs for 72 h (HD1 and WAS4), or with anti-CD3 mAbs plus anti-CD28 mAb–coated beads for 120 h (HD5 and WAS21). Proliferation was measured by [ 3 H]thymidine incorporation (a), and IFN-γ secretion was measured by cytometric bead array (b). Filled bars, activated effector T cells (abbreviated as E); gray bars, activated effector T cells in the presence of nTreg cells isolated from HDs; dashed bars, activated effector T cells in the presence of nTreg cells isolated from WAS patients; dotted bars, activated nTreg cells alone. Percentages of suppression are indicated. (c and d) Cumulative graph of autologous suppression of proliferation (c) and IFN-γ production (d) at the indicated effector T cell/nTreg cell ratio. Dots represent percentage of suppression of each HD or WAS patient, and bars depict the respective median value.

Techniques Used: Isolation, Cell Culture, Irradiation

Thymic generation of nTreg cells in WAS −/− mice. (a) Thymic development of nTreg cells in WT and WAS − / − mice. CD4/CD8 flow cytometric plots were gated on live thymocytes, total Foxp3 + cells, and mature CD24 lo Foxp3 + cells. Numbers indicate the percentage of cells in the respective region. Data are representative of 12 mice per group analyzed in three independent experiments. (b) Immunophenotype of CD4 + single positive thymocytes from representative WT and WAS − / − mice. Numbers indicate the percentages and MFI of CD25 + cells. (c) Percentage, absolute count, and MFI of CD25 + cells among CD4 + single positive thymocytes. Mean ± SD of 13 mice per group are shown. (d) Foxp3 and CD25 expression in CD4 + single positive thymocytes. Numbers indicate the percentage of cells in the respective region. (e) Absolute count of the indicated thymocyte populations. Mean ± SD of 13 mice per group is shown. *, P
Figure Legend Snippet: Thymic generation of nTreg cells in WAS −/− mice. (a) Thymic development of nTreg cells in WT and WAS − / − mice. CD4/CD8 flow cytometric plots were gated on live thymocytes, total Foxp3 + cells, and mature CD24 lo Foxp3 + cells. Numbers indicate the percentage of cells in the respective region. Data are representative of 12 mice per group analyzed in three independent experiments. (b) Immunophenotype of CD4 + single positive thymocytes from representative WT and WAS − / − mice. Numbers indicate the percentages and MFI of CD25 + cells. (c) Percentage, absolute count, and MFI of CD25 + cells among CD4 + single positive thymocytes. Mean ± SD of 13 mice per group are shown. (d) Foxp3 and CD25 expression in CD4 + single positive thymocytes. Numbers indicate the percentage of cells in the respective region. (e) Absolute count of the indicated thymocyte populations. Mean ± SD of 13 mice per group is shown. *, P

Techniques Used: Mouse Assay, Flow Cytometry, Expressing

Allogeneic suppression by human nTreg cells. (a and b) Ability of nTreg cells freshly isolated from WAS patients and HDs to suppress allogeneic CD4 + CD25 − effector T cells from HDs. Effector T cells were cultured in the presence of nTreg cells at the indicated ratio and stimulated with CD3-depleted irradiated allogeneic APCs and anti-CD3 mAbs for 72 h (HD1 and WAS4), anti-CD3 mAbs plus anti-CD28 mAb–coated beads for 72 h (HD2 and WAS14), or anti-CD3 mAbs plus anti-CD28 mAb–coated beads for 120 h (HD4 and WAS17). Proliferation was measured by [ 3 H]thymidine incorporation (a), and IFN-γ secretion was measured by cytometric bead array (b). Filled bars, activated effector T cells (abbreviated as E); gray bars, activated effector T cells in the presence of nTreg cells isolated from HDs; dashed bars, activated effector T cells in the presence of nTreg cells isolated from WAS patients; dotted bars, activated nTreg cells alone. Percentages of suppression are indicated. (c and d) Cumulative graph of allogeneic suppression of proliferation (c) and IFN-γ production (d) at the indicated effector T cell/nTreg cell ratio. Dots represent percentage of suppression of each HD or WAS patient, and bars depict the respective median value. *, P
Figure Legend Snippet: Allogeneic suppression by human nTreg cells. (a and b) Ability of nTreg cells freshly isolated from WAS patients and HDs to suppress allogeneic CD4 + CD25 − effector T cells from HDs. Effector T cells were cultured in the presence of nTreg cells at the indicated ratio and stimulated with CD3-depleted irradiated allogeneic APCs and anti-CD3 mAbs for 72 h (HD1 and WAS4), anti-CD3 mAbs plus anti-CD28 mAb–coated beads for 72 h (HD2 and WAS14), or anti-CD3 mAbs plus anti-CD28 mAb–coated beads for 120 h (HD4 and WAS17). Proliferation was measured by [ 3 H]thymidine incorporation (a), and IFN-γ secretion was measured by cytometric bead array (b). Filled bars, activated effector T cells (abbreviated as E); gray bars, activated effector T cells in the presence of nTreg cells isolated from HDs; dashed bars, activated effector T cells in the presence of nTreg cells isolated from WAS patients; dotted bars, activated nTreg cells alone. Percentages of suppression are indicated. (c and d) Cumulative graph of allogeneic suppression of proliferation (c) and IFN-γ production (d) at the indicated effector T cell/nTreg cell ratio. Dots represent percentage of suppression of each HD or WAS patient, and bars depict the respective median value. *, P

Techniques Used: Isolation, Cell Culture, Irradiation

TCR-mediated activation of WAS −/− nTreg cells. (a) Proliferation of WT and WAS − / − nTreg cells stimulated with 10 μg/ml of plate-bound anti-CD3 mAbs, 100 U/ml IL-2, and/or 1 μg/ml anti-CD28 mAbs as indicated. The average cpm ± SEM of three independent experiments is shown. (b) TGF-β production by WT and WAS − / − nTreg cells activated as in a. The average concentration ± SEM of three independent experiments is shown. (c) TCR down-regulation in nTreg cells and CD4 + CD25 − effector T cells after stimulation with anti-CD3 mAbs. Plotted values represent the average percentage of CD3 + cells of three mice per group. Error bars represent SD. For nTreg cells, *, P
Figure Legend Snippet: TCR-mediated activation of WAS −/− nTreg cells. (a) Proliferation of WT and WAS − / − nTreg cells stimulated with 10 μg/ml of plate-bound anti-CD3 mAbs, 100 U/ml IL-2, and/or 1 μg/ml anti-CD28 mAbs as indicated. The average cpm ± SEM of three independent experiments is shown. (b) TGF-β production by WT and WAS − / − nTreg cells activated as in a. The average concentration ± SEM of three independent experiments is shown. (c) TCR down-regulation in nTreg cells and CD4 + CD25 − effector T cells after stimulation with anti-CD3 mAbs. Plotted values represent the average percentage of CD3 + cells of three mice per group. Error bars represent SD. For nTreg cells, *, P

Techniques Used: Activation Assay, Concentration Assay, Mouse Assay

Cell count and immunophenotype of nTreg cells in the spleens of WAS −/− mice. (a) Immunophenotype of CD4 + splenocytes from representative WT and WAS − / − mice. Numbers indicate the percentages and MFI of CD25 + cells. (b) Percentage, absolute count, and MFI of CD25 + cells among CD4 + splenocytes. Mean ± SD of 13 mice per group is shown. *, P
Figure Legend Snippet: Cell count and immunophenotype of nTreg cells in the spleens of WAS −/− mice. (a) Immunophenotype of CD4 + splenocytes from representative WT and WAS − / − mice. Numbers indicate the percentages and MFI of CD25 + cells. (b) Percentage, absolute count, and MFI of CD25 + cells among CD4 + splenocytes. Mean ± SD of 13 mice per group is shown. *, P

Techniques Used: Cell Counting, Mouse Assay

Suppressor activity of WAS −/− nTreg cells in vitro and in vivo. (a) In vitro suppression of WT or WAS − / − CD90 + CD25 − effector T cells (abbreviated as WT E and WAS − / − E, respectively). Effector T cells were mixed with purified CD4 + CD25 + nTreg cells isolated from WT or WAS − / − mice at the indicated ratios and stimulated with 10 μg/ml of plate-bound anti-CD3 mAbs. Median proliferation of effector T cells: WT = 26,380 cpm and WAS − / − = 6,360 cpm. Mean percentage of suppression ± SEM of the indicated number of independent experiments is shown. *, P
Figure Legend Snippet: Suppressor activity of WAS −/− nTreg cells in vitro and in vivo. (a) In vitro suppression of WT or WAS − / − CD90 + CD25 − effector T cells (abbreviated as WT E and WAS − / − E, respectively). Effector T cells were mixed with purified CD4 + CD25 + nTreg cells isolated from WT or WAS − / − mice at the indicated ratios and stimulated with 10 μg/ml of plate-bound anti-CD3 mAbs. Median proliferation of effector T cells: WT = 26,380 cpm and WAS − / − = 6,360 cpm. Mean percentage of suppression ± SEM of the indicated number of independent experiments is shown. *, P

Techniques Used: Activity Assay, In Vitro, In Vivo, Purification, Isolation, Mouse Assay

WASP expression in murine nTreg cells. (a) WASP expression in CD4 + CD25 + , CD4 + CD25 − , and CD8 + T cells. Splenocytes from WT mice were stained with anti-WASP mAbs (filled histogram) or isotype matched control antibodies (empty histogram) and gated on the indicated T cell subset. Numbers indicate percentages and MFI of WASP + cells. (b) F-actin and WASP localization in murine splenic WT CD4 + CD25 − and CD4 + CD25 + T cells upon stimulation. CD4 + CD25 − effector T cells (left) and CD4 + CD25 + nTreg cells (right) were stimulated with WT APCs (stained in orange) in the absence or presence of 10 μg/ml anti-CD3 mAbs. Images showing F-actin staining (left column), WASP staining (middle column), and their bright field overlay (right column) are shown. These images are representative of at least 100 T cell–APC conjugates that were evaluated for each experimental condition. White arrows indicate F-actin and WASP polarization at the T cell–APC interface. (c) Percentage of WT CD4 + CD25 + nTreg cells and WT CD4 + CD25 − effector T cells showing F-actin polarization at the T cell–APC interface in the presence of APCs and in the absence or presence of the indicated amount of anti-CD3 mAbs. Histogram bars represent the average percentage (± SD) of F-actin polarization in T cells from three independent experiments. *, P
Figure Legend Snippet: WASP expression in murine nTreg cells. (a) WASP expression in CD4 + CD25 + , CD4 + CD25 − , and CD8 + T cells. Splenocytes from WT mice were stained with anti-WASP mAbs (filled histogram) or isotype matched control antibodies (empty histogram) and gated on the indicated T cell subset. Numbers indicate percentages and MFI of WASP + cells. (b) F-actin and WASP localization in murine splenic WT CD4 + CD25 − and CD4 + CD25 + T cells upon stimulation. CD4 + CD25 − effector T cells (left) and CD4 + CD25 + nTreg cells (right) were stimulated with WT APCs (stained in orange) in the absence or presence of 10 μg/ml anti-CD3 mAbs. Images showing F-actin staining (left column), WASP staining (middle column), and their bright field overlay (right column) are shown. These images are representative of at least 100 T cell–APC conjugates that were evaluated for each experimental condition. White arrows indicate F-actin and WASP polarization at the T cell–APC interface. (c) Percentage of WT CD4 + CD25 + nTreg cells and WT CD4 + CD25 − effector T cells showing F-actin polarization at the T cell–APC interface in the presence of APCs and in the absence or presence of the indicated amount of anti-CD3 mAbs. Histogram bars represent the average percentage (± SD) of F-actin polarization in T cells from three independent experiments. *, P

Techniques Used: Expressing, Mouse Assay, Staining

Immunophenotype of nTreg cells from human peripheral blood. (a) CD4 + FOXP3 + cells in the peripheral blood of WAS patients. PBMCs from four representative HDs and seven WAS patients (WAS) were stained with anti-CD4 and anti-FOXP3 mAbs. Results shown are gated on the CD4 + T cells. Numbers indicate the percentages of FOXP3 + cells among CD4 + T cells. (b) Percentage of FOXP3 + cells (left), CD25 + FOXP3 + cells (middle), and CD25 − FOXP3 + cells (right) among CD4 + T cells of HDs ( n = 16) and WAS patients ( n = 7). Bars represent the median value for each group.
Figure Legend Snippet: Immunophenotype of nTreg cells from human peripheral blood. (a) CD4 + FOXP3 + cells in the peripheral blood of WAS patients. PBMCs from four representative HDs and seven WAS patients (WAS) were stained with anti-CD4 and anti-FOXP3 mAbs. Results shown are gated on the CD4 + T cells. Numbers indicate the percentages of FOXP3 + cells among CD4 + T cells. (b) Percentage of FOXP3 + cells (left), CD25 + FOXP3 + cells (middle), and CD25 − FOXP3 + cells (right) among CD4 + T cells of HDs ( n = 16) and WAS patients ( n = 7). Bars represent the median value for each group.

Techniques Used: Staining

2) Product Images from "Differential Requirement for the CD45 Splicing Regulator hnRNPLL for Accumulation of NKT and Conventional T Cells"

Article Title: Differential Requirement for the CD45 Splicing Regulator hnRNPLL for Accumulation of NKT and Conventional T Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0026440

Normal numbers of NKT cell in the thymus and periphery of Hnrpll thu/thu mice despite of reduced number of conventional T cells in the periphery. (A) Graphs show absolute number of CD4 and CD8 conventional T cells in wild type (+/+) and Hnrpll thu/thu (thu/thu) mice (n = 9 for wild type and n = 8 for Hnrpll thu/thu analysed in two independent experiments). Bars represent the mean ± s.d. values. (B) Representative flow cytometric dot plots of NKT cells (α-GalCer-loaded CD1d tetramer + TCRβ + ) in the thymus (Thy) and spleen (Spl) in wild type (+/+) and Hnrpll thu/thu (thu/thu) mice. (C) Graph shows absolute number of NKT cells in the different tissues of wild type (+/+) and Hnrpll thu/thu (thu/thu) mice (n = 12 for wild type and n = 11 for Hnrpll thu/thu analysed in three independent experiments). Bars represent the mean ± s.d. values. (D) Graph shows the ratio of absolute number of NKT cells to absolute number of TCRβ + conventional T cells (n = 12 for wild type (+/+) and n = 11 for Hnrpll thu/thu (thu/thu) analysed in three independent experiments). Bars represent the mean ± s.d. values. (E) Representative flow cytometric dot plots comparing the expression of CD4 versus TCRβ on CD1d-tetramer + TCRβ + NKT cells in the thymus, and spleen of wild type (+/+) and Hnrpll thu/thu (thu/thu) mice. (F) Graph shows absolute number of CD4 + and CD4 − NKT cell subsets in the thymus, spleen and liver of individual wild type (+/+) and Hnrpll thu/thu (thu/thu) mice (n = 9 for wild type and n = 8 for Hnrpll thu/thu analysed in two independent experiments). Bars represent the mean ± s.d. values.
Figure Legend Snippet: Normal numbers of NKT cell in the thymus and periphery of Hnrpll thu/thu mice despite of reduced number of conventional T cells in the periphery. (A) Graphs show absolute number of CD4 and CD8 conventional T cells in wild type (+/+) and Hnrpll thu/thu (thu/thu) mice (n = 9 for wild type and n = 8 for Hnrpll thu/thu analysed in two independent experiments). Bars represent the mean ± s.d. values. (B) Representative flow cytometric dot plots of NKT cells (α-GalCer-loaded CD1d tetramer + TCRβ + ) in the thymus (Thy) and spleen (Spl) in wild type (+/+) and Hnrpll thu/thu (thu/thu) mice. (C) Graph shows absolute number of NKT cells in the different tissues of wild type (+/+) and Hnrpll thu/thu (thu/thu) mice (n = 12 for wild type and n = 11 for Hnrpll thu/thu analysed in three independent experiments). Bars represent the mean ± s.d. values. (D) Graph shows the ratio of absolute number of NKT cells to absolute number of TCRβ + conventional T cells (n = 12 for wild type (+/+) and n = 11 for Hnrpll thu/thu (thu/thu) analysed in three independent experiments). Bars represent the mean ± s.d. values. (E) Representative flow cytometric dot plots comparing the expression of CD4 versus TCRβ on CD1d-tetramer + TCRβ + NKT cells in the thymus, and spleen of wild type (+/+) and Hnrpll thu/thu (thu/thu) mice. (F) Graph shows absolute number of CD4 + and CD4 − NKT cell subsets in the thymus, spleen and liver of individual wild type (+/+) and Hnrpll thu/thu (thu/thu) mice (n = 9 for wild type and n = 8 for Hnrpll thu/thu analysed in two independent experiments). Bars represent the mean ± s.d. values.

Techniques Used: Mouse Assay, Flow Cytometry, Expressing

The Hnrpll mutation does not disrupt cytokine production by thymic, splenic and hepatic NKT cells. (A) Representative histograms showing intracellular cytokine production by α-GalCer-CD1d tetramer + TCRβ + NKT cells from wild type (+/+) and Hnrpll thu/thu (thu/thu) mice that were either resting (shaded grey) or following activation with PMA and ionomycin (black line). (B) Frequency of cytokine producing cells of total NKT cells in the thymus of wild type and Hnrpll thu/thu mice. (C) Frequency of cytokine producing cells of total NKT cells in the spleen of wild type and Hnrpll thu/thu mice. (D) Frequency of cytokine producing cells of total NKT cells in the liver of wild type and Hnrpll thu/thu mice. Bars represent the mean value of each group ± s.d. from one of two independent experiments with a group of n = 3–5 mice per group in each.
Figure Legend Snippet: The Hnrpll mutation does not disrupt cytokine production by thymic, splenic and hepatic NKT cells. (A) Representative histograms showing intracellular cytokine production by α-GalCer-CD1d tetramer + TCRβ + NKT cells from wild type (+/+) and Hnrpll thu/thu (thu/thu) mice that were either resting (shaded grey) or following activation with PMA and ionomycin (black line). (B) Frequency of cytokine producing cells of total NKT cells in the thymus of wild type and Hnrpll thu/thu mice. (C) Frequency of cytokine producing cells of total NKT cells in the spleen of wild type and Hnrpll thu/thu mice. (D) Frequency of cytokine producing cells of total NKT cells in the liver of wild type and Hnrpll thu/thu mice. Bars represent the mean value of each group ± s.d. from one of two independent experiments with a group of n = 3–5 mice per group in each.

Techniques Used: Mutagenesis, Mouse Assay, Activation Assay

Decreased NK1.1 during NKT cell development in Hnrpll thu/thu mice. (A, C) Representative flow cytometric dot plots showing the development stages of NKT cell in the (a) thymus and (c) spleen in intact wild type (+/+) and Hnrpll thu/thu (thu/thu) mice based on expression of CD44 and NK1.1 that were gated on α-Galcer-Cd1d tetramer + TCRβ + NKT cells. (B, D) Graphs show the absolute number of the three development stages of NKT cell in the (b) thymus and (d) spleen and liver respectively (n = 9 for wild type and n = 8 for Hnrpll thu/thu analysed in two independent experiments). Bars represent the mean ± s.d. values. (E, F) Graphs show the geometric mean florescence intensity (MFI) of NK1.1 in the thymic and splenic NKT cells respectively (Data are representative of three independent experiments with n = 3–5 mice per group in each). Bars represent the mean ± s.d. values. (G) Graph shows the absolute number of NK1.1 + TCRβ − NK cell in the spleen (Data are representative of three independent experiments with n = 3–5 mice per group in each). Bars represent the mean ± s.d. values. (H) Graph shows the geometric mean florescence intensity (MFI) of NK1.1 in the splenic NK cells (Data are representative of three independent experiments with n = 3–5 mice per group in each). Bars represent the mean ± s.d. values. (I) Representative overlay histograms comparing the expression of CD1d on DP thymocytes and B220 + cells in the spleen. (J) Representative overlay histograms showing the intracellular expression of T-Bet in NKT cells from the thymus of a wild type (dotted line) and Hnrpll thu/thu mouse (solid line) (Data are representative of two independent experiments with n = 3–5 mice per group in each).
Figure Legend Snippet: Decreased NK1.1 during NKT cell development in Hnrpll thu/thu mice. (A, C) Representative flow cytometric dot plots showing the development stages of NKT cell in the (a) thymus and (c) spleen in intact wild type (+/+) and Hnrpll thu/thu (thu/thu) mice based on expression of CD44 and NK1.1 that were gated on α-Galcer-Cd1d tetramer + TCRβ + NKT cells. (B, D) Graphs show the absolute number of the three development stages of NKT cell in the (b) thymus and (d) spleen and liver respectively (n = 9 for wild type and n = 8 for Hnrpll thu/thu analysed in two independent experiments). Bars represent the mean ± s.d. values. (E, F) Graphs show the geometric mean florescence intensity (MFI) of NK1.1 in the thymic and splenic NKT cells respectively (Data are representative of three independent experiments with n = 3–5 mice per group in each). Bars represent the mean ± s.d. values. (G) Graph shows the absolute number of NK1.1 + TCRβ − NK cell in the spleen (Data are representative of three independent experiments with n = 3–5 mice per group in each). Bars represent the mean ± s.d. values. (H) Graph shows the geometric mean florescence intensity (MFI) of NK1.1 in the splenic NK cells (Data are representative of three independent experiments with n = 3–5 mice per group in each). Bars represent the mean ± s.d. values. (I) Representative overlay histograms comparing the expression of CD1d on DP thymocytes and B220 + cells in the spleen. (J) Representative overlay histograms showing the intracellular expression of T-Bet in NKT cells from the thymus of a wild type (dotted line) and Hnrpll thu/thu mouse (solid line) (Data are representative of two independent experiments with n = 3–5 mice per group in each).

Techniques Used: Mouse Assay, Flow Cytometry, Expressing

3) Product Images from "Hsp65-Producing Lactococcus lactis Prevents Inflammatory Intestinal Disease in Mice by IL-10- and TLR2-Dependent Pathways"

Article Title: Hsp65-Producing Lactococcus lactis Prevents Inflammatory Intestinal Disease in Mice by IL-10- and TLR2-Dependent Pathways

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00030

Hsp65-producing L. lactis (Hsp65-LL) induces increased frequencies of regulatory T cells (Tregs) in mice . C57BL/6 mice were pretreated or not (naïve) with medium (CT), empty vector-harboring L. lactis (CT-LL), or Hsp65-LL for 4 days and colitis was induced 10 days later by 1.5% DSS in drinking water. Seventy-two hours after DSS administration, mice were sacrificed and mesenteric lymph nodes (mLNs) removed. Cells were stained with fluorescein isothiocyanate-conjugated (FITC)-anti-CD4, PE-anti-Foxp3, Bio-CD25 and CY5.5-STV (A) or with FITC-anti-CD4, PE-anti-CD25, Bio-LAP, and Cy5.5-STV (B) . Alternatively, C57BL/6 mice were orally pretreated or not (naïve) with Hsp65-LL during 4 days. Either 4 or 10 days after the last day of oral treatment frequencies of Treg populations were analyzed by flow cytometry. (C) Spleen and (D) mLN cells were stained with Cy5.5 anti-CD4 and PE anti-Foxp3. (E) Spleen cells were stained with Cy5.5 anti-CD4; biotin anti-LAP, and APC streptavidin. CD4 + Foxp3 + and CD4 + LAP + cells were gated on lymphocyte population ( N = 4). Results are representative of three independent experiments. Bar graphs are shown as mean ± SEM. Analysis of variance, Tukey’s post hoc test, p
Figure Legend Snippet: Hsp65-producing L. lactis (Hsp65-LL) induces increased frequencies of regulatory T cells (Tregs) in mice . C57BL/6 mice were pretreated or not (naïve) with medium (CT), empty vector-harboring L. lactis (CT-LL), or Hsp65-LL for 4 days and colitis was induced 10 days later by 1.5% DSS in drinking water. Seventy-two hours after DSS administration, mice were sacrificed and mesenteric lymph nodes (mLNs) removed. Cells were stained with fluorescein isothiocyanate-conjugated (FITC)-anti-CD4, PE-anti-Foxp3, Bio-CD25 and CY5.5-STV (A) or with FITC-anti-CD4, PE-anti-CD25, Bio-LAP, and Cy5.5-STV (B) . Alternatively, C57BL/6 mice were orally pretreated or not (naïve) with Hsp65-LL during 4 days. Either 4 or 10 days after the last day of oral treatment frequencies of Treg populations were analyzed by flow cytometry. (C) Spleen and (D) mLN cells were stained with Cy5.5 anti-CD4 and PE anti-Foxp3. (E) Spleen cells were stained with Cy5.5 anti-CD4; biotin anti-LAP, and APC streptavidin. CD4 + Foxp3 + and CD4 + LAP + cells were gated on lymphocyte population ( N = 4). Results are representative of three independent experiments. Bar graphs are shown as mean ± SEM. Analysis of variance, Tukey’s post hoc test, p

Techniques Used: Mouse Assay, Plasmid Preparation, Staining, Flow Cytometry, Cytometry

Anti-colitogenic effect of Hsp65-producing L. lactis (Hsp65-LL) is dependent on IL-10 . Groups of 6-week-old IL-10-deficient (IL-10 −/− ) mice or control (129Sv/Ev) mice were pretreated or not (naïve) with medium (CT) or Hsp65-LL for four consecutive days. Ten days later, mice were given 1.5% DSS in drinking water for 7 days. (A) Macroscopic score including body weight loss, stool consistency, and bleeding were scored at day 7. (B) At day 7, colons were collected and dissected for histological analysis by hematoxylin and eosin staining. Histological scores (sum of loss of mucosal architecture, cellular infiltration, muscle thickening, crypt abscess formation, and goblet cell depletion) were ranked blindly ( N = 4). (C,D) Groups of 6-week-old IL-10-deficient mice were pretreated or not (naïve) with medium (CT), empty vector-harboring L. lactis (CT-LL), or Hsp65-LL for four consecutive days. When IL-10 −/− mice completed 12 weeks of age, macroscopic and histological scores were ranked. (E) Cytokines such as IFN-γ, IL-17, and IL-10 were measured in colon extracts at the end of the experiment. (F) Lamina propria (LP) T cells from entire colon, cells isolated from mesenteric lymph nodes (mLNs) or from cecal lymph nodes (cLN) were obtained from IL-10 −/− mice. Frequency of CD4 + CD25 + LAP + T cells was assessed by flow cytometry and gated in the lymphocyte population ( N = 8). Results are representatives of two independent experiments. Bar graphs are shown as mean ± SEM. Analysis of variance, Tukey’s post hoc test, p
Figure Legend Snippet: Anti-colitogenic effect of Hsp65-producing L. lactis (Hsp65-LL) is dependent on IL-10 . Groups of 6-week-old IL-10-deficient (IL-10 −/− ) mice or control (129Sv/Ev) mice were pretreated or not (naïve) with medium (CT) or Hsp65-LL for four consecutive days. Ten days later, mice were given 1.5% DSS in drinking water for 7 days. (A) Macroscopic score including body weight loss, stool consistency, and bleeding were scored at day 7. (B) At day 7, colons were collected and dissected for histological analysis by hematoxylin and eosin staining. Histological scores (sum of loss of mucosal architecture, cellular infiltration, muscle thickening, crypt abscess formation, and goblet cell depletion) were ranked blindly ( N = 4). (C,D) Groups of 6-week-old IL-10-deficient mice were pretreated or not (naïve) with medium (CT), empty vector-harboring L. lactis (CT-LL), or Hsp65-LL for four consecutive days. When IL-10 −/− mice completed 12 weeks of age, macroscopic and histological scores were ranked. (E) Cytokines such as IFN-γ, IL-17, and IL-10 were measured in colon extracts at the end of the experiment. (F) Lamina propria (LP) T cells from entire colon, cells isolated from mesenteric lymph nodes (mLNs) or from cecal lymph nodes (cLN) were obtained from IL-10 −/− mice. Frequency of CD4 + CD25 + LAP + T cells was assessed by flow cytometry and gated in the lymphocyte population ( N = 8). Results are representatives of two independent experiments. Bar graphs are shown as mean ± SEM. Analysis of variance, Tukey’s post hoc test, p

Techniques Used: Mouse Assay, Staining, Plasmid Preparation, Isolation, Flow Cytometry, Cytometry

4) Product Images from "Calcineurin-mediated IL-2 production by CD11chighMHCII+ myeloid cells is crucial for intestinal immune homeostasis"

Article Title: Calcineurin-mediated IL-2 production by CD11chighMHCII+ myeloid cells is crucial for intestinal immune homeostasis

Journal: Nature Communications

doi: 10.1038/s41467-018-03495-3

Systemic and intestinal immune homeostasis in Il2 CD11c and Il2 CD4 mice. a RBC count and b total number of splenocytes and proportion of CD4 + T cells, FoxP3 + Treg cells, and B220 + B cells in spleens of Il2 fl / fl , Il2 CD11c , and Il2 CD4 mice. Representative images of spleens are shown. Data represent the means ± standard error of 2–6 experiments ( n = 2–3 mice/group per experiment, 6–13 weeks old). ** P
Figure Legend Snippet: Systemic and intestinal immune homeostasis in Il2 CD11c and Il2 CD4 mice. a RBC count and b total number of splenocytes and proportion of CD4 + T cells, FoxP3 + Treg cells, and B220 + B cells in spleens of Il2 fl / fl , Il2 CD11c , and Il2 CD4 mice. Representative images of spleens are shown. Data represent the means ± standard error of 2–6 experiments ( n = 2–3 mice/group per experiment, 6–13 weeks old). ** P

Techniques Used: Mouse Assay

Il2 or Cnb1 deficiency in DCs causes a dysregulated T-cell response. a , b Naïve congenic OTII cells were sorted from spleens of donor mice and transferred intravenously into Rag2 KO Il2 fl / fl and Rag2 KO Il2 CD11c ( a ) or Cnb1 CD11c and Cnb1 fl / fl mice ( b ). After 24 h, mice were administered OVA protein antigen by daily oral gavage for 5 days. The frequencies of CD4 + T cells expressing FoxP3, IL-17, and IFNγ were examined in the mesenteric lymph node 7 days after adoptive cell transfer. Representative FACS plots of OTII donor CD4 + T cells are shown (left panels). Data represent the means ± standard deviation of two experiments ( n = 1–2 mice/group). * P
Figure Legend Snippet: Il2 or Cnb1 deficiency in DCs causes a dysregulated T-cell response. a , b Naïve congenic OTII cells were sorted from spleens of donor mice and transferred intravenously into Rag2 KO Il2 fl / fl and Rag2 KO Il2 CD11c ( a ) or Cnb1 CD11c and Cnb1 fl / fl mice ( b ). After 24 h, mice were administered OVA protein antigen by daily oral gavage for 5 days. The frequencies of CD4 + T cells expressing FoxP3, IL-17, and IFNγ were examined in the mesenteric lymph node 7 days after adoptive cell transfer. Representative FACS plots of OTII donor CD4 + T cells are shown (left panels). Data represent the means ± standard deviation of two experiments ( n = 1–2 mice/group). * P

Techniques Used: Mouse Assay, Expressing, FACS, Standard Deviation

DC-derived IL-2 suppresses pathogenic CD4 + T-cell expansion in the intestine. a Change in body weight and b severity of colitis based on colon length of Il2 CD11c and Il2 fl / fl , Rag2 knockout (KO) mice ( Rag2 KO IL - 2 fl / fl and Rag2 KO IL - 2 CD11c mice) adoptively transferred with naïve CD4 + T cells either alone or in combination with Treg cells isolated from spleens of C57BL/6 mice. Representative pictures of colons after adoptive T-cell transfer are shown. Data represent the means ± standard error of two experiments ( n = 2–5 mice/group per experiment). ** P
Figure Legend Snippet: DC-derived IL-2 suppresses pathogenic CD4 + T-cell expansion in the intestine. a Change in body weight and b severity of colitis based on colon length of Il2 CD11c and Il2 fl / fl , Rag2 knockout (KO) mice ( Rag2 KO IL - 2 fl / fl and Rag2 KO IL - 2 CD11c mice) adoptively transferred with naïve CD4 + T cells either alone or in combination with Treg cells isolated from spleens of C57BL/6 mice. Representative pictures of colons after adoptive T-cell transfer are shown. Data represent the means ± standard error of two experiments ( n = 2–5 mice/group per experiment). ** P

Techniques Used: Derivative Assay, Knock-Out, Mouse Assay, Isolation

Calcineurin B and NFAT expression in mouse intestinal myeloid cells. a Relative expression levels of Cnb1 , Nfat1 , and Nfat2 mRNAs in intestinal CD11c high MHCII + cells (CD11b + and CD11b − ) and MLN CD3 + T cells, assessed by qRT-PCR. Data represent the means ± standard error of three experiments ( n ≥ 7 mice/exp). b , c Percentage of NFAT-1 + cells ( b ) and NFAT-1 protein levels ( c ) in CD11c high MHCII + cells (CD11b + and CD11b − ) obtained from spleen, MLN, PP, and colonic LP (LP-colon), assessed by flow cytometry. Splenic CD3 + CD4 + T cells are included for comparison. Data represent the means ± standard error of three experiments ( n = 4–5 mice/experiment). ** P
Figure Legend Snippet: Calcineurin B and NFAT expression in mouse intestinal myeloid cells. a Relative expression levels of Cnb1 , Nfat1 , and Nfat2 mRNAs in intestinal CD11c high MHCII + cells (CD11b + and CD11b − ) and MLN CD3 + T cells, assessed by qRT-PCR. Data represent the means ± standard error of three experiments ( n ≥ 7 mice/exp). b , c Percentage of NFAT-1 + cells ( b ) and NFAT-1 protein levels ( c ) in CD11c high MHCII + cells (CD11b + and CD11b − ) obtained from spleen, MLN, PP, and colonic LP (LP-colon), assessed by flow cytometry. Splenic CD3 + CD4 + T cells are included for comparison. Data represent the means ± standard error of three experiments ( n = 4–5 mice/experiment). ** P

Techniques Used: Expressing, Quantitative RT-PCR, Mouse Assay, Flow Cytometry, Cytometry

5) Product Images from "ABNORMAL REGULATORY AND EFFECTOR T CELL FUNCTION PREDISPOSE TO AUTOIMMUNITY FOLLOWING XENOGENEIC THYMIC TRANSPLANTATION 1"

Article Title: ABNORMAL REGULATORY AND EFFECTOR T CELL FUNCTION PREDISPOSE TO AUTOIMMUNITY FOLLOWING XENOGENEIC THYMIC TRANSPLANTATION 1

Journal:

doi:

Co-transfer of CD4 or CD8 normal BALB/c splenocytes (nSPL) protects against autoimmune disease induced by adoptive transfer of splenocytes from FPG nude mice (GSPL)
Figure Legend Snippet: Co-transfer of CD4 or CD8 normal BALB/c splenocytes (nSPL) protects against autoimmune disease induced by adoptive transfer of splenocytes from FPG nude mice (GSPL)

Techniques Used: Adoptive Transfer Assay, Mouse Assay

6) Product Images from "Early Detection of T cell Transfer-induced Autoimmune Colitis by In Vivo Imaging System"

Article Title: Early Detection of T cell Transfer-induced Autoimmune Colitis by In Vivo Imaging System

Journal: Scientific Reports

doi: 10.1038/srep35635

Luc-expressing T cell expanded in PBLs and cytokine production in sera. ( a ) Massive expansion of Luc-expressing T cell in PBLs. At D28 PAT, PBLs were collected and stained as described in Methods. Stained PBLs were then analyzed by flow cytometry. Percentages of CD4 + T cells to total PBLs and percentages of CD4 + Foxp3 + CD25 + T cells to total CD4 + T cells are shown. ( b ) Sera were collected at D14 and D35 PAT, and the serum cytokines as indicated were determined by CBA as described in Methods. Data from four repeated experiments are combined (naïve T, n = 14; naïve T + T reg , n = 14) and represented by Means ± SEM. ** p
Figure Legend Snippet: Luc-expressing T cell expanded in PBLs and cytokine production in sera. ( a ) Massive expansion of Luc-expressing T cell in PBLs. At D28 PAT, PBLs were collected and stained as described in Methods. Stained PBLs were then analyzed by flow cytometry. Percentages of CD4 + T cells to total PBLs and percentages of CD4 + Foxp3 + CD25 + T cells to total CD4 + T cells are shown. ( b ) Sera were collected at D14 and D35 PAT, and the serum cytokines as indicated were determined by CBA as described in Methods. Data from four repeated experiments are combined (naïve T, n = 14; naïve T + T reg , n = 14) and represented by Means ± SEM. ** p

Techniques Used: Expressing, Staining, Flow Cytometry, Cytometry, Crocin Bleaching Assay

DEXA treatment reduced donor T cells survival and induced suppressive cytokine production in sera. ( a ) Reduction of Luc-expressing T cell expansion in PBLs from Rag1-ko mice that received naïve T cells and were treated with DEXA for three weeks. At D28 PAT, PBLs were s stained as described in Methods, and then analyzed by flow cytometry. Percentages of CD4 + T cells in total PBL and percentages of CD4 + Foxp3 + CD25 + T cells in CD4 + T cells are shown. ( b ) Sera were collected at D14 and D35 PAT and analyzed for serum TNF, IFN-γ, IL-6, and IL-17A production by CBA kits as described in Methods. Data from four repeated experiments are combined (DEXA, n = 16; saline, n = 12) and represented by Means ± SEM. (** p
Figure Legend Snippet: DEXA treatment reduced donor T cells survival and induced suppressive cytokine production in sera. ( a ) Reduction of Luc-expressing T cell expansion in PBLs from Rag1-ko mice that received naïve T cells and were treated with DEXA for three weeks. At D28 PAT, PBLs were s stained as described in Methods, and then analyzed by flow cytometry. Percentages of CD4 + T cells in total PBL and percentages of CD4 + Foxp3 + CD25 + T cells in CD4 + T cells are shown. ( b ) Sera were collected at D14 and D35 PAT and analyzed for serum TNF, IFN-γ, IL-6, and IL-17A production by CBA kits as described in Methods. Data from four repeated experiments are combined (DEXA, n = 16; saline, n = 12) and represented by Means ± SEM. (** p

Techniques Used: Expressing, Mouse Assay, Staining, Flow Cytometry, Cytometry, Crocin Bleaching Assay

Early detection of autoimmune colitis by BLI analysis. ( a ) Percentages of CD4 + CD25 − CD45RB hi naïve T cells from Luc Tg. spleens before and after sorting are shown. ( b ) Continued BLI analysis of Luc-expressing, naïve T cell-transferred Rag1-ko mice. Rag1-ko host mice that received Luc-expressing naïve T cells or naïve T cells + CD4 + CD25 + CD45RB lo T reg cells were analyzed with IVIS twice a week. Abdominal BLIs were significantly detected at D12-D15 PAT and thereafter. Total influx at each time point after adoptive transfer is shown. ( c ) Changes of BW of Rag1-ko host mice after transfer of Luc-expressing naïve T cells. BW shown at each time point is normalized by the BW at D0. Data from four repeated experiments are combined (naïve T, n = 14; naïve T + T reg , n = 14) and represented by Means ± SEM. (* p
Figure Legend Snippet: Early detection of autoimmune colitis by BLI analysis. ( a ) Percentages of CD4 + CD25 − CD45RB hi naïve T cells from Luc Tg. spleens before and after sorting are shown. ( b ) Continued BLI analysis of Luc-expressing, naïve T cell-transferred Rag1-ko mice. Rag1-ko host mice that received Luc-expressing naïve T cells or naïve T cells + CD4 + CD25 + CD45RB lo T reg cells were analyzed with IVIS twice a week. Abdominal BLIs were significantly detected at D12-D15 PAT and thereafter. Total influx at each time point after adoptive transfer is shown. ( c ) Changes of BW of Rag1-ko host mice after transfer of Luc-expressing naïve T cells. BW shown at each time point is normalized by the BW at D0. Data from four repeated experiments are combined (naïve T, n = 14; naïve T + T reg , n = 14) and represented by Means ± SEM. (* p

Techniques Used: Expressing, Mouse Assay, Adoptive Transfer Assay

7) Product Images from "Hsp65-Producing Lactococcus lactis Prevents Inflammatory Intestinal Disease in Mice by IL-10- and TLR2-Dependent Pathways"

Article Title: Hsp65-Producing Lactococcus lactis Prevents Inflammatory Intestinal Disease in Mice by IL-10- and TLR2-Dependent Pathways

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00030

Hsp65-producing L. lactis (Hsp65-LL) induces increased frequencies of regulatory T cells (Tregs) in mice . C57BL/6 mice were pretreated or not (naïve) with medium (CT), empty vector-harboring L. lactis (CT-LL), or Hsp65-LL for 4 days and colitis was induced 10 days later by 1.5% DSS in drinking water. Seventy-two hours after DSS administration, mice were sacrificed and mesenteric lymph nodes (mLNs) removed. Cells were stained with fluorescein isothiocyanate-conjugated (FITC)-anti-CD4, PE-anti-Foxp3, Bio-CD25 and CY5.5-STV (A) or with FITC-anti-CD4, PE-anti-CD25, Bio-LAP, and Cy5.5-STV (B) . Alternatively, C57BL/6 mice were orally pretreated or not (naïve) with Hsp65-LL during 4 days. Either 4 or 10 days after the last day of oral treatment frequencies of Treg populations were analyzed by flow cytometry. (C) Spleen and (D) mLN cells were stained with Cy5.5 anti-CD4 and PE anti-Foxp3. (E) Spleen cells were stained with Cy5.5 anti-CD4; biotin anti-LAP, and APC streptavidin. CD4 + Foxp3 + and CD4 + LAP + cells were gated on lymphocyte population ( N = 4). Results are representative of three independent experiments. Bar graphs are shown as mean ± SEM. Analysis of variance, Tukey’s post hoc test, p
Figure Legend Snippet: Hsp65-producing L. lactis (Hsp65-LL) induces increased frequencies of regulatory T cells (Tregs) in mice . C57BL/6 mice were pretreated or not (naïve) with medium (CT), empty vector-harboring L. lactis (CT-LL), or Hsp65-LL for 4 days and colitis was induced 10 days later by 1.5% DSS in drinking water. Seventy-two hours after DSS administration, mice were sacrificed and mesenteric lymph nodes (mLNs) removed. Cells were stained with fluorescein isothiocyanate-conjugated (FITC)-anti-CD4, PE-anti-Foxp3, Bio-CD25 and CY5.5-STV (A) or with FITC-anti-CD4, PE-anti-CD25, Bio-LAP, and Cy5.5-STV (B) . Alternatively, C57BL/6 mice were orally pretreated or not (naïve) with Hsp65-LL during 4 days. Either 4 or 10 days after the last day of oral treatment frequencies of Treg populations were analyzed by flow cytometry. (C) Spleen and (D) mLN cells were stained with Cy5.5 anti-CD4 and PE anti-Foxp3. (E) Spleen cells were stained with Cy5.5 anti-CD4; biotin anti-LAP, and APC streptavidin. CD4 + Foxp3 + and CD4 + LAP + cells were gated on lymphocyte population ( N = 4). Results are representative of three independent experiments. Bar graphs are shown as mean ± SEM. Analysis of variance, Tukey’s post hoc test, p

Techniques Used: Mouse Assay, Plasmid Preparation, Staining, Flow Cytometry, Cytometry

8) Product Images from "T cells that cannot respond to TGF-? escape control by CD4+CD25+ regulatory T cells"

Article Title: T cells that cannot respond to TGF-? escape control by CD4+CD25+ regulatory T cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20040685

Cytokine production by the progeny of transferred CD4 + CD45RB high cells isolated from the LP. RAG-1 −/− mice received CD4 + T cells as described in Fig. 1 . 6–8 wk after transfer, LP cells were isolated and stimulated for 18 h with plate-bound anti-CD3 antibody. (A) Levels of cytokine expression were determined by flow cytometry. Scatter plots are gated on CD4 + CD45.1 + lymphocytes to identify progeny of transferred CD4 + CD45RB high cells and are representative of four to five mice per group. (B) Absolute numbers of cytokine-producing cells were determined by multiplying the frequency of cytokine producing cells after stimulation in vitro by the total number of LP leukocytes. Data are pooled from two independent experiments.
Figure Legend Snippet: Cytokine production by the progeny of transferred CD4 + CD45RB high cells isolated from the LP. RAG-1 −/− mice received CD4 + T cells as described in Fig. 1 . 6–8 wk after transfer, LP cells were isolated and stimulated for 18 h with plate-bound anti-CD3 antibody. (A) Levels of cytokine expression were determined by flow cytometry. Scatter plots are gated on CD4 + CD45.1 + lymphocytes to identify progeny of transferred CD4 + CD45RB high cells and are representative of four to five mice per group. (B) Absolute numbers of cytokine-producing cells were determined by multiplying the frequency of cytokine producing cells after stimulation in vitro by the total number of LP leukocytes. Data are pooled from two independent experiments.

Techniques Used: Isolation, Mouse Assay, Expressing, Flow Cytometry, Cytometry, In Vitro

Frequency of KJ-1.26 + cells is similar among CD4 + CD25 + cells from either DO11.10 or DO11.10 TGF-β1 −/− mice. Mice analyzed at 6–8 wk of age. CD4 enriched splenocytes from either DO11.10 or DO11.10 TGF-β1 −/− mice were analyzed by flow cytometry for the expression of CD4 and CD25. Numbers indicate percentage of CD25 + cells among CD4 + cells. CD4 + CD25 + cells were further analyzed for the expression of the clonotypic TCR by mAb KJ-1.26. Numbers indicate percentage KJ-1.26 + cells among CD4 + CD25 + cells. Data are representative of six mice per group.
Figure Legend Snippet: Frequency of KJ-1.26 + cells is similar among CD4 + CD25 + cells from either DO11.10 or DO11.10 TGF-β1 −/− mice. Mice analyzed at 6–8 wk of age. CD4 enriched splenocytes from either DO11.10 or DO11.10 TGF-β1 −/− mice were analyzed by flow cytometry for the expression of CD4 and CD25. Numbers indicate percentage of CD25 + cells among CD4 + cells. CD4 + CD25 + cells were further analyzed for the expression of the clonotypic TCR by mAb KJ-1.26. Numbers indicate percentage KJ-1.26 + cells among CD4 + CD25 + cells. Data are representative of six mice per group.

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Expressing

Suppression of colitis by thymic but not splenic dnTβRII CD4 + CD25 + cells. RAG-1 −/− mice received 2–4 × 10 5 CD4 + CD45RB high cells alone or in combination with 1.5–2 × 10 5 CD4 + CD25 + cells isolated from the spleen or thymus of either WT or dnTβRII Tg mice. In addition, some mice received splenic CD4 + CD25 + cells alone. Mice were killed 6–8 wk after transfer, and colons were taken for histological analysis. Data show colitis scores for individual mice taken from three independent experiments.
Figure Legend Snippet: Suppression of colitis by thymic but not splenic dnTβRII CD4 + CD25 + cells. RAG-1 −/− mice received 2–4 × 10 5 CD4 + CD45RB high cells alone or in combination with 1.5–2 × 10 5 CD4 + CD25 + cells isolated from the spleen or thymus of either WT or dnTβRII Tg mice. In addition, some mice received splenic CD4 + CD25 + cells alone. Mice were killed 6–8 wk after transfer, and colons were taken for histological analysis. Data show colitis scores for individual mice taken from three independent experiments.

Techniques Used: Mouse Assay, Isolation

CD4 + CD25 + cells from dnTβRII mice express normal levels of FoxP3. (A) CD4 + CD25 + and CD4 + CD25 − cells were isolated from spleens of WT (white bars) and dnTβRII (black bars) mice. mRNA was recovered and analyzed for expression of FoxP3 and CD3γ by quantitative real-time PCR. Data show FoxP3 mRNA expression normalized to CD3γ levels as mean ± SEM for triplicate wells. A second experiment gave similar results. (B) Tissue sections from spleens of WT and dnTβRII mice were stained for CD4 (green) and FoxP3 (red). FoxP3 + CD4 + cells show red nuclear staining and green surface staining. Frequency of FoxP3 + cells among CD4 + cells: WT 6.4 ± 1.3% and dnTβRII 6.6 ± 1.1%. Numbers represent mean and SEM from three individual mice per group. Original magnification, 200.
Figure Legend Snippet: CD4 + CD25 + cells from dnTβRII mice express normal levels of FoxP3. (A) CD4 + CD25 + and CD4 + CD25 − cells were isolated from spleens of WT (white bars) and dnTβRII (black bars) mice. mRNA was recovered and analyzed for expression of FoxP3 and CD3γ by quantitative real-time PCR. Data show FoxP3 mRNA expression normalized to CD3γ levels as mean ± SEM for triplicate wells. A second experiment gave similar results. (B) Tissue sections from spleens of WT and dnTβRII mice were stained for CD4 (green) and FoxP3 (red). FoxP3 + CD4 + cells show red nuclear staining and green surface staining. Frequency of FoxP3 + cells among CD4 + cells: WT 6.4 ± 1.3% and dnTβRII 6.6 ± 1.1%. Numbers represent mean and SEM from three individual mice per group. Original magnification, 200.

Techniques Used: Mouse Assay, Isolation, Expressing, Real-time Polymerase Chain Reaction, Staining

CD4 + CD25 + T cells are unable to prevent colitis induced by transfer of dnTβRII CD4 + CD45RB high cells. RAG-1 −/− mice received 2 × 10 5 CD4 + CD45RB high cells isolated from either WT or dnTβRII mice. In addition, some mice also received an equivalent number of WT CD4 + CD25 + T cells. (A) Data show the mean weight of six animals per group and are representative of three independent experiments. Numbers in parentheses indicate number of animals with clinical signs of disease killed at day 45 and weights for these animals are included in the mean weights. CD4 + CD25 + cells confer significant protection from wasting induced by WT CD4 + CD45RB high cells (P
Figure Legend Snippet: CD4 + CD25 + T cells are unable to prevent colitis induced by transfer of dnTβRII CD4 + CD45RB high cells. RAG-1 −/− mice received 2 × 10 5 CD4 + CD45RB high cells isolated from either WT or dnTβRII mice. In addition, some mice also received an equivalent number of WT CD4 + CD25 + T cells. (A) Data show the mean weight of six animals per group and are representative of three independent experiments. Numbers in parentheses indicate number of animals with clinical signs of disease killed at day 45 and weights for these animals are included in the mean weights. CD4 + CD25 + cells confer significant protection from wasting induced by WT CD4 + CD45RB high cells (P

Techniques Used: Mouse Assay, Isolation

CD4 + CD25 + T cells from DO11.10.TGF-β1 −/− mice retain the ability to suppress colitis. C.B-17 SCID mice received 4 × 10 5 CD4 + CD45RB high cells alone or in combination with 10 5 CD4 + CD25 + cells isolated from 6–8-wk-old WT, DO11.10, or DO11.10.TGF-β1 −/− mice. Some mice received anti–TGF-β mAb. (A) Incidence and severity of colitis at time of sacrifice. (n) Number of mice in each group. Data are pooled from five independent experiments. CD4 + CD25 + cells from WT, DO11.10, and DO11.10 TGF-β1 −/− mice confer significant protection from colitis (P
Figure Legend Snippet: CD4 + CD25 + T cells from DO11.10.TGF-β1 −/− mice retain the ability to suppress colitis. C.B-17 SCID mice received 4 × 10 5 CD4 + CD45RB high cells alone or in combination with 10 5 CD4 + CD25 + cells isolated from 6–8-wk-old WT, DO11.10, or DO11.10.TGF-β1 −/− mice. Some mice received anti–TGF-β mAb. (A) Incidence and severity of colitis at time of sacrifice. (n) Number of mice in each group. Data are pooled from five independent experiments. CD4 + CD25 + cells from WT, DO11.10, and DO11.10 TGF-β1 −/− mice confer significant protection from colitis (P

Techniques Used: Mouse Assay, Isolation

9) Product Images from "Expanded B cell population blocks regulatory T cells and exacerbates ileitis in a murine model of Crohn disease"

Article Title: Expanded B cell population blocks regulatory T cells and exacerbates ileitis in a murine model of Crohn disease

Journal: Journal of Clinical Investigation

doi: 10.1172/JCI200420855

Soluble IgA production increased in SAMP1/YitFc versus AKR mice. ( A ) Representative dot plots of κ (1:100 dilution, FL2) and λ (1:1, FL1) light chain antibody isotype levels within MLN supernatants from AKR mice ( n = 2) and SAMP1/YitFc mice ( n = 2), measured with a cytometric bead array containing isotype-specific beads with preset FL3 intensities. MLNs were crushed, resuspended in 5 ml, and centrifuged to remove the cell pellet from the tested supernatants. An increase of at least twofold in IgG1 κ, IgA κ, IgM κ, IgA λ, and IgM λ was seen in SAMP1/YitFc versus AKR MLNs, as measured by mean fluorescence intensity of the isotype-specific beads. ( B ) ELISA detecting IgA antibody concentrations (mean ± SEM) in serum samples collected via cardiac puncture from SAMP1/YitFc mice ( n = 12) and wild-type AKR mice ( n = 13). ( C ) Concentrations of IgA ( n = 4), IgM ( n = 2), and IgG2a ( n = 2), measured in triplicate by ELISA, from 3-, 7-, and 11-day anti-CD3–stimulated cocultures of SAMP1/YitFc CD4 + T cells and B cells versus AKR CD4 + T cells and B cells (105 T cells/well and 105 B cells/well). *Significantly greater than AKR concentrations ( P
Figure Legend Snippet: Soluble IgA production increased in SAMP1/YitFc versus AKR mice. ( A ) Representative dot plots of κ (1:100 dilution, FL2) and λ (1:1, FL1) light chain antibody isotype levels within MLN supernatants from AKR mice ( n = 2) and SAMP1/YitFc mice ( n = 2), measured with a cytometric bead array containing isotype-specific beads with preset FL3 intensities. MLNs were crushed, resuspended in 5 ml, and centrifuged to remove the cell pellet from the tested supernatants. An increase of at least twofold in IgG1 κ, IgA κ, IgM κ, IgA λ, and IgM λ was seen in SAMP1/YitFc versus AKR MLNs, as measured by mean fluorescence intensity of the isotype-specific beads. ( B ) ELISA detecting IgA antibody concentrations (mean ± SEM) in serum samples collected via cardiac puncture from SAMP1/YitFc mice ( n = 12) and wild-type AKR mice ( n = 13). ( C ) Concentrations of IgA ( n = 4), IgM ( n = 2), and IgG2a ( n = 2), measured in triplicate by ELISA, from 3-, 7-, and 11-day anti-CD3–stimulated cocultures of SAMP1/YitFc CD4 + T cells and B cells versus AKR CD4 + T cells and B cells (105 T cells/well and 105 B cells/well). *Significantly greater than AKR concentrations ( P

Techniques Used: Mouse Assay, Fluorescence, Enzyme-linked Immunosorbent Assay

SAMP1/YitFc MLN and ileum contain large populations of IgA + cells. ( A ) SAMP1/YitFc MLN (top) contain considerably more IgA-secreting cells (dark brown spots) and soluble IgA (diffuse light brown) than do AKR MLN (bottom), as shown by immunostaining of paraffin-embedded MLN sections. ( B ) Left, representative dot plots of B220 versus IgA expression, with quadrant percentages (mean ± SEM), demonstrating an increase in mature B cells (B220hi) and plasmablasts (B220int) expressing IgA in SAMP1/YitFc MLNs ( n = 13) versus AKR MLNs ( n = 6). *Significantly greater ( P
Figure Legend Snippet: SAMP1/YitFc MLN and ileum contain large populations of IgA + cells. ( A ) SAMP1/YitFc MLN (top) contain considerably more IgA-secreting cells (dark brown spots) and soluble IgA (diffuse light brown) than do AKR MLN (bottom), as shown by immunostaining of paraffin-embedded MLN sections. ( B ) Left, representative dot plots of B220 versus IgA expression, with quadrant percentages (mean ± SEM), demonstrating an increase in mature B cells (B220hi) and plasmablasts (B220int) expressing IgA in SAMP1/YitFc MLNs ( n = 13) versus AKR MLNs ( n = 6). *Significantly greater ( P

Techniques Used: Immunostaining, Expressing

10) Product Images from "IL-10-producing CD4+ T cells negatively regulate fucosylation of epithelial cells in the gut"

Article Title: IL-10-producing CD4+ T cells negatively regulate fucosylation of epithelial cells in the gut

Journal: Scientific Reports

doi: 10.1038/srep15918

αβT cells regulate fucosylation of ileal ECs. ( A ) Whole-mount staining of the ileum of C57BL/6, Tcrb −/− , and Tcrd −/− mice was performed with UEA-1 (red) and wheat germ agglutinin (green). Scale bars: 100 μm. Data are representative of 4 independent experiments. ( B,C ) Ileal ECs were stained with CD45 antibody and UEA-1. CD45 − cells were gated and analyzed for UEA-1 binding by flow cytometry. Representative dot plots are shown in ( B ). Mean percentages of UEA-1 + ECs are shown ± s.d. (n = 6) in ( C ). ( D ) Quantitative PCR analysis of Fut2 expression in ileal ECs. Data normalized against the expression of Gapdh are shown as mean ± s.d. ( n = 6 from 2 independent experiments).
Figure Legend Snippet: αβT cells regulate fucosylation of ileal ECs. ( A ) Whole-mount staining of the ileum of C57BL/6, Tcrb −/− , and Tcrd −/− mice was performed with UEA-1 (red) and wheat germ agglutinin (green). Scale bars: 100 μm. Data are representative of 4 independent experiments. ( B,C ) Ileal ECs were stained with CD45 antibody and UEA-1. CD45 − cells were gated and analyzed for UEA-1 binding by flow cytometry. Representative dot plots are shown in ( B ). Mean percentages of UEA-1 + ECs are shown ± s.d. (n = 6) in ( C ). ( D ) Quantitative PCR analysis of Fut2 expression in ileal ECs. Data normalized against the expression of Gapdh are shown as mean ± s.d. ( n = 6 from 2 independent experiments).

Techniques Used: Staining, Mouse Assay, Binding Assay, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Expressing

T cell deficiency enhances epithelial fucosylation in the ileum. ( A ) Whole-mount staining of the ileum of C57BL/6, Rag1 −/− , Tcrb −/− Tcrd −/− , and Igh-6 −/− mice was performed with UEA-1 (red) for α-1,2 fucose staining and wheat germ agglutinin (green) for epithelial cell counterstaining. Scale bars: 100 μm. Data are representative of 4 independent experiments. (B,C) Ileal ECs were stained with CD45 antibody and UEA-1. CD45 − cells were gated and analyzed for UEA-1 binding by flow cytometry. Representative dot plots are shown in ( B ). Mean percentages ± s.d. ( n = 6) of UEA-1 + ECs are shown in ( C ). ( D ) Quantitative PCR analysis of Fut2 expression in ileal ECs. Data normalized against the expression of Gapdh are shown as mean ± s.d. ( n = 6 from 2 independent experiments).
Figure Legend Snippet: T cell deficiency enhances epithelial fucosylation in the ileum. ( A ) Whole-mount staining of the ileum of C57BL/6, Rag1 −/− , Tcrb −/− Tcrd −/− , and Igh-6 −/− mice was performed with UEA-1 (red) for α-1,2 fucose staining and wheat germ agglutinin (green) for epithelial cell counterstaining. Scale bars: 100 μm. Data are representative of 4 independent experiments. (B,C) Ileal ECs were stained with CD45 antibody and UEA-1. CD45 − cells were gated and analyzed for UEA-1 binding by flow cytometry. Representative dot plots are shown in ( B ). Mean percentages ± s.d. ( n = 6) of UEA-1 + ECs are shown in ( C ). ( D ) Quantitative PCR analysis of Fut2 expression in ileal ECs. Data normalized against the expression of Gapdh are shown as mean ± s.d. ( n = 6 from 2 independent experiments).

Techniques Used: Staining, Mouse Assay, Binding Assay, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Expressing

11) Product Images from "WASP regulates suppressor activity of human and murine CD4+CD25+FOXP3+ natural regulatory T cells"

Article Title: WASP regulates suppressor activity of human and murine CD4+CD25+FOXP3+ natural regulatory T cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20061334

Thymic generation of nTreg cells in WAS −/− mice. (a) Thymic development of nTreg cells in WT and WAS − / − mice. CD4/CD8 flow cytometric plots were gated on live thymocytes, total Foxp3 + cells, and mature CD24 lo Foxp3 + cells. Numbers indicate the percentage of cells in the respective region. Data are representative of 12 mice per group analyzed in three independent experiments. (b) Immunophenotype of CD4 + single positive thymocytes from representative WT and WAS − / − mice. Numbers indicate the percentages and MFI of CD25 + cells. (c) Percentage, absolute count, and MFI of CD25 + cells among CD4 + single positive thymocytes. Mean ± SD of 13 mice per group are shown. (d) Foxp3 and CD25 expression in CD4 + single positive thymocytes. Numbers indicate the percentage of cells in the respective region. (e) Absolute count of the indicated thymocyte populations. Mean ± SD of 13 mice per group is shown. *, P
Figure Legend Snippet: Thymic generation of nTreg cells in WAS −/− mice. (a) Thymic development of nTreg cells in WT and WAS − / − mice. CD4/CD8 flow cytometric plots were gated on live thymocytes, total Foxp3 + cells, and mature CD24 lo Foxp3 + cells. Numbers indicate the percentage of cells in the respective region. Data are representative of 12 mice per group analyzed in three independent experiments. (b) Immunophenotype of CD4 + single positive thymocytes from representative WT and WAS − / − mice. Numbers indicate the percentages and MFI of CD25 + cells. (c) Percentage, absolute count, and MFI of CD25 + cells among CD4 + single positive thymocytes. Mean ± SD of 13 mice per group are shown. (d) Foxp3 and CD25 expression in CD4 + single positive thymocytes. Numbers indicate the percentage of cells in the respective region. (e) Absolute count of the indicated thymocyte populations. Mean ± SD of 13 mice per group is shown. *, P

Techniques Used: Mouse Assay, Flow Cytometry, Expressing

WASP expression in murine nTreg cells. (a) WASP expression in CD4 + CD25 + , CD4 + CD25 − , and CD8 + T cells. Splenocytes from WT mice were stained with anti-WASP mAbs (filled histogram) or isotype matched control antibodies (empty histogram) and gated on the indicated T cell subset. Numbers indicate percentages and MFI of WASP + cells. (b) F-actin and WASP localization in murine splenic WT CD4 + CD25 − and CD4 + CD25 + T cells upon stimulation. CD4 + CD25 − effector T cells (left) and CD4 + CD25 + nTreg cells (right) were stimulated with WT APCs (stained in orange) in the absence or presence of 10 μg/ml anti-CD3 mAbs. Images showing F-actin staining (left column), WASP staining (middle column), and their bright field overlay (right column) are shown. These images are representative of at least 100 T cell–APC conjugates that were evaluated for each experimental condition. White arrows indicate F-actin and WASP polarization at the T cell–APC interface. (c) Percentage of WT CD4 + CD25 + nTreg cells and WT CD4 + CD25 − effector T cells showing F-actin polarization at the T cell–APC interface in the presence of APCs and in the absence or presence of the indicated amount of anti-CD3 mAbs. Histogram bars represent the average percentage (± SD) of F-actin polarization in T cells from three independent experiments. *, P
Figure Legend Snippet: WASP expression in murine nTreg cells. (a) WASP expression in CD4 + CD25 + , CD4 + CD25 − , and CD8 + T cells. Splenocytes from WT mice were stained with anti-WASP mAbs (filled histogram) or isotype matched control antibodies (empty histogram) and gated on the indicated T cell subset. Numbers indicate percentages and MFI of WASP + cells. (b) F-actin and WASP localization in murine splenic WT CD4 + CD25 − and CD4 + CD25 + T cells upon stimulation. CD4 + CD25 − effector T cells (left) and CD4 + CD25 + nTreg cells (right) were stimulated with WT APCs (stained in orange) in the absence or presence of 10 μg/ml anti-CD3 mAbs. Images showing F-actin staining (left column), WASP staining (middle column), and their bright field overlay (right column) are shown. These images are representative of at least 100 T cell–APC conjugates that were evaluated for each experimental condition. White arrows indicate F-actin and WASP polarization at the T cell–APC interface. (c) Percentage of WT CD4 + CD25 + nTreg cells and WT CD4 + CD25 − effector T cells showing F-actin polarization at the T cell–APC interface in the presence of APCs and in the absence or presence of the indicated amount of anti-CD3 mAbs. Histogram bars represent the average percentage (± SD) of F-actin polarization in T cells from three independent experiments. *, P

Techniques Used: Expressing, Mouse Assay, Staining

12) Product Images from "The survival of memory CD8 T cells that is mediated by IL-15 correlates with sustained protection against malaria survival of memory CD8 T cells that is mediated by IL-15 correlates with sustained protection against malaria"

Article Title: The survival of memory CD8 T cells that is mediated by IL-15 correlates with sustained protection against malaria survival of memory CD8 T cells that is mediated by IL-15 correlates with sustained protection against malaria

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

doi: 10.4049/jimmunol.1203396

IL-15KO CD8 T cells respond to antigen from Pb γ-spz Wt and IL-15KO mice (3–5 mice/group) were immunized with 3 weekly doses of Pb γ-spz followed one week later by infectious sporozoite challenge as described in materials and methods. (A) Livers from naïve, immune, or immune/challenged (CH) mice were isolated 1 week after last exposure to antigen and stained with anti-CD3, -CD8, -CD44, -CD62L mAbs to determine the number of total CD8 T cells, CD8 T E/EM cells and CD8 T CM cells. The results show the number of cells as the mean ± SD (B) IHMC were isolated from wt and IL-15KO mice one week following the 3 rd immunization with Pb γ-spz. IFN-γ was detected by cytokine secretion assay, as described in Materials and Methods. CD8 T cells were enriched by magnetic bead separation and CD44 hi CD45RB hi or CD44 hi CD45RB lo T cells were identified by flow cytometry. The results are representative of several experiments performed under the same conditions and show the number of IFN-γ secreting CD8 T CM cells and CD8 T E/EM cells per 10 6 IHMC determined for each individual mouse and are presented as the mean ± SD, *p
Figure Legend Snippet: IL-15KO CD8 T cells respond to antigen from Pb γ-spz Wt and IL-15KO mice (3–5 mice/group) were immunized with 3 weekly doses of Pb γ-spz followed one week later by infectious sporozoite challenge as described in materials and methods. (A) Livers from naïve, immune, or immune/challenged (CH) mice were isolated 1 week after last exposure to antigen and stained with anti-CD3, -CD8, -CD44, -CD62L mAbs to determine the number of total CD8 T cells, CD8 T E/EM cells and CD8 T CM cells. The results show the number of cells as the mean ± SD (B) IHMC were isolated from wt and IL-15KO mice one week following the 3 rd immunization with Pb γ-spz. IFN-γ was detected by cytokine secretion assay, as described in Materials and Methods. CD8 T cells were enriched by magnetic bead separation and CD44 hi CD45RB hi or CD44 hi CD45RB lo T cells were identified by flow cytometry. The results are representative of several experiments performed under the same conditions and show the number of IFN-γ secreting CD8 T CM cells and CD8 T E/EM cells per 10 6 IHMC determined for each individual mouse and are presented as the mean ± SD, *p

Techniques Used: Mouse Assay, Isolation, Staining, Flow Cytometry, Cytometry

CD8 T CM cells, having elevated expression of IL-15Rβ (CD122), are the primary cells responding to IL-15 IHMC were isolated from C57BL/6 mice (3 per group) 1 month after a tertiary immunization with Pb γ-spz. (A) CD62L + and CD62L − T cells were separated by magnetic bead isolation procedure; a second round of isolation using CD8 magnetic beads resulted in the following subpopulations: CD8 + CD62L + and CD8 + CD62L − T cells as well as CD8 − CD62L + and CD8 − CD62L − T cells. Each T cell subset was cultured at 4 × 10 5 cells/0.2 ml culture medium for 96 hours in the presence of the indicted concentrations of IL-15; during the last 16hrs of culture, 1μCi of 3 H-TdR was added to each well. Results are presented as the mean CPM ± SD of 3 H-TdR uptake in triplicate wells and are representative of 3 separate experiments. (B) Liver CD8 T cells, isolated by negative magnetic beads selection, were stained with CFSE and incubated for 7 days in the presence of 100 ng/ml IL-15, or 100ng/ml IL-2. Harvested cells were stained with mAbs against CD8, CD44 and CD45RB. Lymphocytes were gated on a forward-side scatter plot, and gates were applied to identify CD8 + T cells that expressed either CD44 hi CD45RB hi (T CM cells) or CD44 hi CD45RB lo (T E/EM cells) phenotypes. Histogram plots represent CFSE dilution with gray shaded area representing CD8 T CM cells and solid contour line representing CD8 T E/EM cells. These results represent one of two separate experiments. (C) Liver CD8 T cells were cultured in the presence of either 100 ng/ml IL-15, or medium alone. Cells were harvested on day 1, 3, and 7 and stained with mAbs against CD44 and CD45RB as described above, fixed, permeabilized and stained with mAbs against Bcl-2. Mean fluorescent intensity (determined for each individual mouse and is presented as the mean ± SD) of Bcl-2 expression of T CM or T E/EM . Bcl-2 determinations on CD8 T CM and CD8 T E/EM cells prior to culture showed MFI=189± 45 for CD8 T CM and MFI= 76± 35 for CD8 T E/EM . Results are representative of 3 separate experiments. *p
Figure Legend Snippet: CD8 T CM cells, having elevated expression of IL-15Rβ (CD122), are the primary cells responding to IL-15 IHMC were isolated from C57BL/6 mice (3 per group) 1 month after a tertiary immunization with Pb γ-spz. (A) CD62L + and CD62L − T cells were separated by magnetic bead isolation procedure; a second round of isolation using CD8 magnetic beads resulted in the following subpopulations: CD8 + CD62L + and CD8 + CD62L − T cells as well as CD8 − CD62L + and CD8 − CD62L − T cells. Each T cell subset was cultured at 4 × 10 5 cells/0.2 ml culture medium for 96 hours in the presence of the indicted concentrations of IL-15; during the last 16hrs of culture, 1μCi of 3 H-TdR was added to each well. Results are presented as the mean CPM ± SD of 3 H-TdR uptake in triplicate wells and are representative of 3 separate experiments. (B) Liver CD8 T cells, isolated by negative magnetic beads selection, were stained with CFSE and incubated for 7 days in the presence of 100 ng/ml IL-15, or 100ng/ml IL-2. Harvested cells were stained with mAbs against CD8, CD44 and CD45RB. Lymphocytes were gated on a forward-side scatter plot, and gates were applied to identify CD8 + T cells that expressed either CD44 hi CD45RB hi (T CM cells) or CD44 hi CD45RB lo (T E/EM cells) phenotypes. Histogram plots represent CFSE dilution with gray shaded area representing CD8 T CM cells and solid contour line representing CD8 T E/EM cells. These results represent one of two separate experiments. (C) Liver CD8 T cells were cultured in the presence of either 100 ng/ml IL-15, or medium alone. Cells were harvested on day 1, 3, and 7 and stained with mAbs against CD44 and CD45RB as described above, fixed, permeabilized and stained with mAbs against Bcl-2. Mean fluorescent intensity (determined for each individual mouse and is presented as the mean ± SD) of Bcl-2 expression of T CM or T E/EM . Bcl-2 determinations on CD8 T CM and CD8 T E/EM cells prior to culture showed MFI=189± 45 for CD8 T CM and MFI= 76± 35 for CD8 T E/EM . Results are representative of 3 separate experiments. *p

Techniques Used: Expressing, Isolation, Mouse Assay, Magnetic Beads, Cell Culture, Selection, Staining, Incubation

13) Product Images from "WASP regulates suppressor activity of human and murine CD4+CD25+FOXP3+ natural regulatory T cells"

Article Title: WASP regulates suppressor activity of human and murine CD4+CD25+FOXP3+ natural regulatory T cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20061334

Thymic generation of nTreg cells in WAS −/− mice. (a) Thymic development of nTreg cells in WT and WAS − / − mice. CD4/CD8 flow cytometric plots were gated on live thymocytes, total Foxp3 + cells, and mature CD24 lo Foxp3 + cells. Numbers indicate the percentage of cells in the respective region. Data are representative of 12 mice per group analyzed in three independent experiments. (b) Immunophenotype of CD4 + single positive thymocytes from representative WT and WAS − / − mice. Numbers indicate the percentages and MFI of CD25 + cells. (c) Percentage, absolute count, and MFI of CD25 + cells among CD4 + single positive thymocytes. Mean ± SD of 13 mice per group are shown. (d) Foxp3 and CD25 expression in CD4 + single positive thymocytes. Numbers indicate the percentage of cells in the respective region. (e) Absolute count of the indicated thymocyte populations. Mean ± SD of 13 mice per group is shown. *, P
Figure Legend Snippet: Thymic generation of nTreg cells in WAS −/− mice. (a) Thymic development of nTreg cells in WT and WAS − / − mice. CD4/CD8 flow cytometric plots were gated on live thymocytes, total Foxp3 + cells, and mature CD24 lo Foxp3 + cells. Numbers indicate the percentage of cells in the respective region. Data are representative of 12 mice per group analyzed in three independent experiments. (b) Immunophenotype of CD4 + single positive thymocytes from representative WT and WAS − / − mice. Numbers indicate the percentages and MFI of CD25 + cells. (c) Percentage, absolute count, and MFI of CD25 + cells among CD4 + single positive thymocytes. Mean ± SD of 13 mice per group are shown. (d) Foxp3 and CD25 expression in CD4 + single positive thymocytes. Numbers indicate the percentage of cells in the respective region. (e) Absolute count of the indicated thymocyte populations. Mean ± SD of 13 mice per group is shown. *, P

Techniques Used: Mouse Assay, Flow Cytometry, Expressing

14) Product Images from "Egr2-independent, Klf1-mediated induction of PD-L1 in CD4+ T cells"

Article Title: Egr2-independent, Klf1-mediated induction of PD-L1 in CD4+ T cells

Journal: Scientific Reports

doi: 10.1038/s41598-018-25302-1

Egr2-independent induction of PD-L1 expression in CD4 + T cells by ectopic expression of Klf1. (a) Venn diagram representing the genomic overlap between LAG3 + Tregs specific transcription factors from microarray analysis of CD4 + T cell subsets from ArrayExpress database (E-MEXP-1343) and PD-L1-inducible candidate genes predicted by JASPAR database. (b) Gene expression levels of Klf1 in CD4 + T cells. Klf1 gene expression in indicated T cell subsets relative to unstimulated naïve CD4 + cells using micro array data set as in (a) (left) and relative to Actb mRNA of each indicated T cell subset confirmed by qRT-PCR (right). * p
Figure Legend Snippet: Egr2-independent induction of PD-L1 expression in CD4 + T cells by ectopic expression of Klf1. (a) Venn diagram representing the genomic overlap between LAG3 + Tregs specific transcription factors from microarray analysis of CD4 + T cell subsets from ArrayExpress database (E-MEXP-1343) and PD-L1-inducible candidate genes predicted by JASPAR database. (b) Gene expression levels of Klf1 in CD4 + T cells. Klf1 gene expression in indicated T cell subsets relative to unstimulated naïve CD4 + cells using micro array data set as in (a) (left) and relative to Actb mRNA of each indicated T cell subset confirmed by qRT-PCR (right). * p

Techniques Used: Expressing, Microarray, Quantitative RT-PCR

Egr2-mediated PD-L1 induction in CD4 + T cells. (a) LAG3 expression in splenocytes from C57BL/6 (B6) mice was analyzed by flow cytometry (FCM). CD25 + Tregs: CD4 + CD25 + T cells; naïve T: CD4 + CD25 − CD45RB high T cells; LAG3 + Tregs: CD4 + CD25 − CD45RB low LAG3 + T cells. (b) PD-L1 expression in T cell subsets. Histograms are gated on CD4 + T cells (left). Data are representative of three independent experiments. Mean fluorescence intensity (MFI) of the indicated T cell subsets is shown (right). (n = 3 per group). * p
Figure Legend Snippet: Egr2-mediated PD-L1 induction in CD4 + T cells. (a) LAG3 expression in splenocytes from C57BL/6 (B6) mice was analyzed by flow cytometry (FCM). CD25 + Tregs: CD4 + CD25 + T cells; naïve T: CD4 + CD25 − CD45RB high T cells; LAG3 + Tregs: CD4 + CD25 − CD45RB low LAG3 + T cells. (b) PD-L1 expression in T cell subsets. Histograms are gated on CD4 + T cells (left). Data are representative of three independent experiments. Mean fluorescence intensity (MFI) of the indicated T cell subsets is shown (right). (n = 3 per group). * p

Techniques Used: Expressing, Mouse Assay, Flow Cytometry, Cytometry, Fluorescence

15) Product Images from "Enhanced susceptibility to chemically induced colitis caused by excessive endosomal TLR signaling in LRBA-deficient mice"

Article Title: Enhanced susceptibility to chemically induced colitis caused by excessive endosomal TLR signaling in LRBA-deficient mice

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1901407116

DCs promote DSS-induced colitis in Lrba −/− mice. ( A – F ) BM transplantation was performed using mice of the indicated genotypes (donor > recipient). ( A and C ) Body weight (% relative to weight on day 0) on day 10 ( A ) or on the indicated day ( C ) after initiation of DSS treatment. Lrba +/+ > Lrba +/+ ( n = 5), Lrba −/− > Lrba +/+ ( n = 4), Lrba +/+ > Lrba −/− ( n = 3), Lrba −/− > Lrba −/− ( n = 5) for A. Lrba +/+ Rag2 −/− > Rag2 −/− ( n = 4), Lrba −/− Rag2 −/− > Rag2 −/− ( n = 4) for C. ( B and D ) DAI and ( E ) colon length on day 10 after initiation of DSS treatment. ( F ) Colon sections stained with hematoxylin and eosin on day 7 after initiation of DSS treatment. (Scale bars, 80 μm.) ( G and H ) CD4 + T cell transfer model of colitis. Naive CD4 + T cells from donor mice were transferred to recipient mice (donor > recipient). ( G ) Body weight (% relative to weight on day 0) on the indicated day after initiation of DSS treatment. C57BL/6J > Lrba +/+ Rag2 −/− ( n = 5), C57BL/6J > Lrba −/− Rag2 −/− ( n = 5). ( H ) DAI on day 16 after naive CD4 + T cell transfer. ( I , Left ) Flow cytometry of colon lamina propria cells from Lrba +/+ and Lrba −/− mice, assessing expression of CTLA4 in CD3 + CD4 + FoxP3 + cell population. ( I , Right ) Quantification of CTLA4 protein expression in colon lamina propria CD3 + CD4 + FoxP3 + cells (mean fluorescence intensity normalized to wild type). ( J – M ) Adoptive transfer of in vitro differentiated BMDCs ( J and K ) or BMpDCs ( L and M ) from donor mice to recipient mice (donor > recipient). ( J and L ) Body weight (% relative to weight on day 0) on the indicated day after initiation of DSS treatment. Lrba +/+ BMDCs > Lrba +/+ ( n = 5), Lrba −/− BMDCs > Lrba +/+ ( n = 4) for J and K . Lrba +/+ BMpDCs > Lrba +/+ ( n = 5), Lrba −/− BMpDCs > Lrba +/+ ( n = 5) for L and M . ( K and M ) DAI on day 10 after initiation of DSS treatment. ( N ) Flow cytometry of peripheral blood cells from BM chimeric mice (donor > recipient) before and after diphtheria toxin (DT) treatment (three doses, day −4, −2, and 2 relative to DSS initiation), assessing expression of CD11c and MHC II. Numbers above boxed regions represent percent cells in each. ( O ) Body weight (% relative to weight on day 0) and ( P ) DAI on day 10 after initiation of DSS treatment of BM chimeric mice (donor > recipient) treated with DT to ablate DCs. All mice were treated with DT. Each symbol ( A , B , D , E , H , I , K , M , O , and P ) represents an individual mouse. * P
Figure Legend Snippet: DCs promote DSS-induced colitis in Lrba −/− mice. ( A – F ) BM transplantation was performed using mice of the indicated genotypes (donor > recipient). ( A and C ) Body weight (% relative to weight on day 0) on day 10 ( A ) or on the indicated day ( C ) after initiation of DSS treatment. Lrba +/+ > Lrba +/+ ( n = 5), Lrba −/− > Lrba +/+ ( n = 4), Lrba +/+ > Lrba −/− ( n = 3), Lrba −/− > Lrba −/− ( n = 5) for A. Lrba +/+ Rag2 −/− > Rag2 −/− ( n = 4), Lrba −/− Rag2 −/− > Rag2 −/− ( n = 4) for C. ( B and D ) DAI and ( E ) colon length on day 10 after initiation of DSS treatment. ( F ) Colon sections stained with hematoxylin and eosin on day 7 after initiation of DSS treatment. (Scale bars, 80 μm.) ( G and H ) CD4 + T cell transfer model of colitis. Naive CD4 + T cells from donor mice were transferred to recipient mice (donor > recipient). ( G ) Body weight (% relative to weight on day 0) on the indicated day after initiation of DSS treatment. C57BL/6J > Lrba +/+ Rag2 −/− ( n = 5), C57BL/6J > Lrba −/− Rag2 −/− ( n = 5). ( H ) DAI on day 16 after naive CD4 + T cell transfer. ( I , Left ) Flow cytometry of colon lamina propria cells from Lrba +/+ and Lrba −/− mice, assessing expression of CTLA4 in CD3 + CD4 + FoxP3 + cell population. ( I , Right ) Quantification of CTLA4 protein expression in colon lamina propria CD3 + CD4 + FoxP3 + cells (mean fluorescence intensity normalized to wild type). ( J – M ) Adoptive transfer of in vitro differentiated BMDCs ( J and K ) or BMpDCs ( L and M ) from donor mice to recipient mice (donor > recipient). ( J and L ) Body weight (% relative to weight on day 0) on the indicated day after initiation of DSS treatment. Lrba +/+ BMDCs > Lrba +/+ ( n = 5), Lrba −/− BMDCs > Lrba +/+ ( n = 4) for J and K . Lrba +/+ BMpDCs > Lrba +/+ ( n = 5), Lrba −/− BMpDCs > Lrba +/+ ( n = 5) for L and M . ( K and M ) DAI on day 10 after initiation of DSS treatment. ( N ) Flow cytometry of peripheral blood cells from BM chimeric mice (donor > recipient) before and after diphtheria toxin (DT) treatment (three doses, day −4, −2, and 2 relative to DSS initiation), assessing expression of CD11c and MHC II. Numbers above boxed regions represent percent cells in each. ( O ) Body weight (% relative to weight on day 0) and ( P ) DAI on day 10 after initiation of DSS treatment of BM chimeric mice (donor > recipient) treated with DT to ablate DCs. All mice were treated with DT. Each symbol ( A , B , D , E , H , I , K , M , O , and P ) represents an individual mouse. * P

Techniques Used: Mouse Assay, Transplantation Assay, Staining, Flow Cytometry, Cytometry, Expressing, Fluorescence, Adoptive Transfer Assay, In Vitro

16) Product Images from "Distinct Roles for CXCR6+ and CXCR6− CD4+ T Cells in the Pathogenesis of Chronic Colitis"

Article Title: Distinct Roles for CXCR6+ and CXCR6− CD4+ T Cells in the Pathogenesis of Chronic Colitis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0065488

CXCR6 expression is not required for development of transfer colitis. ( A ) Body weight of  Rag1 −/−  recipients of i.v. injected purified CD45RB high CD4 +  T cells form  Cxcr6 +/Egfp  or  Cxcr6 Egfp/Egfp  (CXCR6-deficient) mice on day 0, presented as percent of original weight. ( B ) Colon weight of the mice in ( A ) on week 7. Data are representative of two independent experiments (mean and s.d.). ( C ) Histology of colon tissues from the mice in  B . ( D ) CXCR6 expression by LP CD4 +  T cells was analyzed by flow cytometry using CXCL16-hFc at 7-week post transfer. ( E ) Expression levels of indicated cytokines in distal colon were analyzed by Q-PCR at 7 weeks after the transfer. Data were normalized to expression of  Gapdh . ( n =  4 or 5; mean and s.d.).
Figure Legend Snippet: CXCR6 expression is not required for development of transfer colitis. ( A ) Body weight of Rag1 −/− recipients of i.v. injected purified CD45RB high CD4 + T cells form Cxcr6 +/Egfp or Cxcr6 Egfp/Egfp (CXCR6-deficient) mice on day 0, presented as percent of original weight. ( B ) Colon weight of the mice in ( A ) on week 7. Data are representative of two independent experiments (mean and s.d.). ( C ) Histology of colon tissues from the mice in B . ( D ) CXCR6 expression by LP CD4 + T cells was analyzed by flow cytometry using CXCL16-hFc at 7-week post transfer. ( E ) Expression levels of indicated cytokines in distal colon were analyzed by Q-PCR at 7 weeks after the transfer. Data were normalized to expression of Gapdh . ( n =  4 or 5; mean and s.d.).

Techniques Used: Expressing, Injection, Purification, Mouse Assay, Flow Cytometry, Cytometry, Polymerase Chain Reaction

CXCR6 expression is related to the production of Th1 and Th17 cytokines. Intracellular staining for cytokine and transcription factors in CD4 +  T cells was performed 8 weeks after naïve CD4 +  T-cell transfer. ( A, B ) The frequency of IL-2 +  cells and IFN-γ +  cells was analyzed in the CXCR6 −  (upper) and CXCR6 +  (lower) subsets ( A ) and the absolute number of T cells were graphed on the basis of the flow cytometric analysis ( B ). ( C, D ) The frequency of IL-17A +  cells and TNF-α +  cells was analyzed in CXCR6 −  (upper) and CXCR6 +  (lower) subset ( C ) and the numbers were graphed on the basis of flow cytometric analysis ( D ). ( E ) The frequency of IFN-γ +  cells and IL-17A +  cells was analyzed. ( F ) The frequency of T-bet +  cells and RORγt +  cells was analyzed. All data are representative from four independent experiments (mean and s.d.). *,  P
Figure Legend Snippet: CXCR6 expression is related to the production of Th1 and Th17 cytokines. Intracellular staining for cytokine and transcription factors in CD4 + T cells was performed 8 weeks after naïve CD4 + T-cell transfer. ( A, B ) The frequency of IL-2 + cells and IFN-γ + cells was analyzed in the CXCR6 − (upper) and CXCR6 + (lower) subsets ( A ) and the absolute number of T cells were graphed on the basis of the flow cytometric analysis ( B ). ( C, D ) The frequency of IL-17A + cells and TNF-α + cells was analyzed in CXCR6 − (upper) and CXCR6 + (lower) subset ( C ) and the numbers were graphed on the basis of flow cytometric analysis ( D ). ( E ) The frequency of IFN-γ + cells and IL-17A + cells was analyzed. ( F ) The frequency of T-bet + cells and RORγt + cells was analyzed. All data are representative from four independent experiments (mean and s.d.). *, P

Techniques Used: Expressing, Staining, Flow Cytometry

LP CXCR6 −  but not CXCR6 +  cells can transfer wasting and colitis upon retransfer into  Rag1 −/−  recipients. ( A ) Schematic transfer protocol. An equal number of CD25 − CXCR6 −  cells or CD25 − CXCR6 + CD4 +  T cell isolated from colon LP of colitic mice was retransferred into  Rag1 −/−  mice. ( B ) Time course of changes in body weight after retransfer of the two subsets or transfer of CD45RB high  naïve T cells. CXCR6 – transferred  Rag1 −/−  mice manifested progressive body weight loss to a similar extent to CD45RB high -transferred  Rag1 −/−  mice, whereas the cohort that received CXCR6 + cells did not show wasting. Data are expressed as the mean and s.e.m of two independent experiments. *,  P
Figure Legend Snippet: LP CXCR6 − but not CXCR6 + cells can transfer wasting and colitis upon retransfer into Rag1 −/− recipients. ( A ) Schematic transfer protocol. An equal number of CD25 − CXCR6 − cells or CD25 − CXCR6 + CD4 + T cell isolated from colon LP of colitic mice was retransferred into Rag1 −/− mice. ( B ) Time course of changes in body weight after retransfer of the two subsets or transfer of CD45RB high naïve T cells. CXCR6 – transferred Rag1 −/− mice manifested progressive body weight loss to a similar extent to CD45RB high -transferred Rag1 −/− mice, whereas the cohort that received CXCR6 + cells did not show wasting. Data are expressed as the mean and s.e.m of two independent experiments. *, P

Techniques Used: Isolation, Mouse Assay

CXCR6 is expressed both by the effector and effector memory CD4 +  T cells in the inflamed colon. LP CXCR6 −  and CXCR6 +  cells were analyzed for expression of activation and memory markers at week 8 post-transfer of naïve CD4 +  T cells. ( A – C ) CD4 +  T cells were gated as CD127 − CD62L − CD27 − CD43 + CD44 +  to measure the proportion of effector T cells (a). ( D – F ) The effector memory population (CD44 + CD127 + ) was subdivided using CD62L and CD27 to measure early effector memory cells (CD62L − CD27 + CD43 + , b) and late effector memory cells (CD62L − CD27 − CD43 + , c). Data are representative of three independent experiments. ( G ) The relative percentages of effector, early effector memory and late effector memory in each subset are shown in a pie chart.
Figure Legend Snippet: CXCR6 is expressed both by the effector and effector memory CD4 + T cells in the inflamed colon. LP CXCR6 − and CXCR6 + cells were analyzed for expression of activation and memory markers at week 8 post-transfer of naïve CD4 + T cells. ( A – C ) CD4 + T cells were gated as CD127 − CD62L − CD27 − CD43 + CD44 + to measure the proportion of effector T cells (a). ( D – F ) The effector memory population (CD44 + CD127 + ) was subdivided using CD62L and CD27 to measure early effector memory cells (CD62L − CD27 + CD43 + , b) and late effector memory cells (CD62L − CD27 − CD43 + , c). Data are representative of three independent experiments. ( G ) The relative percentages of effector, early effector memory and late effector memory in each subset are shown in a pie chart.

Techniques Used: Expressing, Activation Assay

17) Product Images from "Bacteria-triggered CD4+ T Regulatory Cells Suppress Helicobacter hepaticus-induced Colitis"

Article Title: Bacteria-triggered CD4+ T Regulatory Cells Suppress Helicobacter hepaticus-induced Colitis

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20020556

H. hepaticus –infected but not naive RAG KO mice develop intestinal inflammation after reconstitution with CD4 + T cells from IL-10 KO mice. (A) Uninfected or H. hepaticus –infected RAG KO mice were inoculated intravenously with 3 × 10 5 CD4 + cells from the MLNs of 11-wk infected IL-10 KO mice, and intestinal pathology was analyzed 8, 15, 22, and 29 d later (solid bars). Naive and infected RAG KO animals receiving no cells were analyzed in parallel (open bars). Bars represent mean cecal histology scores ± SD of three or four mice per group. *, P
Figure Legend Snippet: H. hepaticus –infected but not naive RAG KO mice develop intestinal inflammation after reconstitution with CD4 + T cells from IL-10 KO mice. (A) Uninfected or H. hepaticus –infected RAG KO mice were inoculated intravenously with 3 × 10 5 CD4 + cells from the MLNs of 11-wk infected IL-10 KO mice, and intestinal pathology was analyzed 8, 15, 22, and 29 d later (solid bars). Naive and infected RAG KO animals receiving no cells were analyzed in parallel (open bars). Bars represent mean cecal histology scores ± SD of three or four mice per group. *, P

Techniques Used: Infection, Mouse Assay

18) Product Images from "Bacteria-triggered CD4+ T Regulatory Cells Suppress Helicobacter hepaticus-induced Colitis"

Article Title: Bacteria-triggered CD4+ T Regulatory Cells Suppress Helicobacter hepaticus-induced Colitis

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20020556

CD25 − CD45RB low and CD25 + CD45RB low CD4 + cells from infected WT mice block colitis via an IL-10–dependent but TGF-β–independent mechanism. Infected RAG KO mice were given either no cells or CD4 + cells from infected IL-10 KO mice and CD25 − CD45RB low or CD25 + CD45RB low CD4 + cells from infected WT mice (3 × 10 5 of each population) and treated with control mAb, anti–IL-10R (both at 1 mg/wk), or anti–TGF-β as indicated. (A) In the first experiment, mice were given 2 mg of anti–TGF-β per wk for 4 wk, (B) and in the second experiment, they were given 4 mg of anti–TGF-β per wk for 2 wk before analysis. •, the cecal histology score from an individual mouse; —, the average for each group. Similar results, although lower histology scores, were seen for the ascending colon where CD25 − cell recipients treated with control or anti–TGF-β mAb displayed indistinguishable scores in B. In A, anti–IL-10R–treated groups had P values of
Figure Legend Snippet: CD25 − CD45RB low and CD25 + CD45RB low CD4 + cells from infected WT mice block colitis via an IL-10–dependent but TGF-β–independent mechanism. Infected RAG KO mice were given either no cells or CD4 + cells from infected IL-10 KO mice and CD25 − CD45RB low or CD25 + CD45RB low CD4 + cells from infected WT mice (3 × 10 5 of each population) and treated with control mAb, anti–IL-10R (both at 1 mg/wk), or anti–TGF-β as indicated. (A) In the first experiment, mice were given 2 mg of anti–TGF-β per wk for 4 wk, (B) and in the second experiment, they were given 4 mg of anti–TGF-β per wk for 2 wk before analysis. •, the cecal histology score from an individual mouse; —, the average for each group. Similar results, although lower histology scores, were seen for the ascending colon where CD25 − cell recipients treated with control or anti–TGF-β mAb displayed indistinguishable scores in B. In A, anti–IL-10R–treated groups had P values of

Techniques Used: Infection, Mouse Assay, Blocking Assay

CD25 − CD45RB low as well as CD25 + CD45RB low CD4 + cells from infected WT mice protect RAG KO mice against colitis. Infected RAG KO mice were given either no cells or 3 × 10 5 CD4 + cells from infected IL-10 KO mice either alone or with 3 × 10 5 CD45RB low CD4 + cells, 3 × 10 5 , 10 5 , or 3.3 × 10 4 CD25 − CD45RB low , or CD25 + CD45RB low CD4 + cells from infected WT mice as indicated. (A) Pathology in the cecum and (B) ascending colon was analyzed 4 wk later. •, an individual mouse; —, the average for each group. Data shown are pooled from two separate experiments. *, P
Figure Legend Snippet: CD25 − CD45RB low as well as CD25 + CD45RB low CD4 + cells from infected WT mice protect RAG KO mice against colitis. Infected RAG KO mice were given either no cells or 3 × 10 5 CD4 + cells from infected IL-10 KO mice either alone or with 3 × 10 5 CD45RB low CD4 + cells, 3 × 10 5 , 10 5 , or 3.3 × 10 4 CD25 − CD45RB low , or CD25 + CD45RB low CD4 + cells from infected WT mice as indicated. (A) Pathology in the cecum and (B) ascending colon was analyzed 4 wk later. •, an individual mouse; —, the average for each group. Data shown are pooled from two separate experiments. *, P

Techniques Used: Infection, Mouse Assay

19) Product Images from "CD44 is involved in selective leucocyte extravasation during inflammatory central nervous system disease"

Article Title: CD44 is involved in selective leucocyte extravasation during inflammatory central nervous system disease

Journal: Immunology

doi: 10.1046/j.1365-2567.1999.00894.x

Anti‐CD44 monoclonal antibody (mAb) IM7 induces the loss of CD44 from lymphocytes in vivo. Animals were injected intraperitoneally (i.p.), daily for 5 days, with 100 µg of either IM7 mAb or rat immunoglobulin. Inguinal lymph nodes were removed 12 hr after the last injection and the level of CD44 on CD4 + T cells was analysed by direct immunofluorescence using CD44‐specific KM201 mAb. The y ‐axis shows cell number and the x ‐axis is mean fluorescence intensity. Background staining gave an intensity of less than 10 fluorescence units.
Figure Legend Snippet: Anti‐CD44 monoclonal antibody (mAb) IM7 induces the loss of CD44 from lymphocytes in vivo. Animals were injected intraperitoneally (i.p.), daily for 5 days, with 100 µg of either IM7 mAb or rat immunoglobulin. Inguinal lymph nodes were removed 12 hr after the last injection and the level of CD44 on CD4 + T cells was analysed by direct immunofluorescence using CD44‐specific KM201 mAb. The y ‐axis shows cell number and the x ‐axis is mean fluorescence intensity. Background staining gave an intensity of less than 10 fluorescence units.

Techniques Used: In Vivo, Injection, Immunofluorescence, Fluorescence, Staining

In vitro shedding of CD44 prevents T‐cell migration into inflammatory central nervous system (CNS) lesions. Following the development of paralysis during acute‐phase chronic‐relapsing experimental allergic encephalomyelitis (CREAE), Biozzi AB/H (Thy1.1 + ) mice were injected intravenously (i.v.) with 1 × 10 7 lymph node cells expressing Thy1.2 antigen, which had been treated with immobilized rat immunoglobulin or CD44‐specific IM7 monoclonal antibody (mAb) for 4 hr at 37°. Animals were perfused 6 hr later and single‐cell suspensions were prepared from the inguinal lymph nodes and spinal cord from each individual animal. Cells were gated to detect lymphocytes, and a dot‐plot was created showing double immunofluorescence flow cytometry of transferred Thy1.2 + T cells (fluorescence intensity on y ‐axis) and the CD4 + T‐cell population (fluorescence intensity on x ‐axis).
Figure Legend Snippet: In vitro shedding of CD44 prevents T‐cell migration into inflammatory central nervous system (CNS) lesions. Following the development of paralysis during acute‐phase chronic‐relapsing experimental allergic encephalomyelitis (CREAE), Biozzi AB/H (Thy1.1 + ) mice were injected intravenously (i.v.) with 1 × 10 7 lymph node cells expressing Thy1.2 antigen, which had been treated with immobilized rat immunoglobulin or CD44‐specific IM7 monoclonal antibody (mAb) for 4 hr at 37°. Animals were perfused 6 hr later and single‐cell suspensions were prepared from the inguinal lymph nodes and spinal cord from each individual animal. Cells were gated to detect lymphocytes, and a dot‐plot was created showing double immunofluorescence flow cytometry of transferred Thy1.2 + T cells (fluorescence intensity on y ‐axis) and the CD4 + T‐cell population (fluorescence intensity on x ‐axis).

Techniques Used: In Vitro, Migration, Mouse Assay, Injection, Expressing, Immunofluorescence, Flow Cytometry, Cytometry, Fluorescence

Anti‐CD44 monoclonal antibody (mAb) IM7 inhibits lymphocyte entry into the central nervous system (CNS). Following the development of chronic‐relapsing experimental allergic encephalomyelitis (CREAE) 13–15 days after the injection of spinal cord homogenate (SCH) in Freund’s complete adjuvant, mice were given five daily injections of phosphate‐buffered saline (PBS) (a) or 100 µg of anti‐CD44 mAb IM7 (b). On day 18 postinjection (p.i.), spinal cords were removed, and sections were made and stained with haematoxylin and eosin. (a) A section from a control mouse with EAE containing large numbers of infiltrating cells. (b) A section from a corresponding area from a clinically well IM7‐treated mouse. Note the lack of infiltrating mononuclear cells.
Figure Legend Snippet: Anti‐CD44 monoclonal antibody (mAb) IM7 inhibits lymphocyte entry into the central nervous system (CNS). Following the development of chronic‐relapsing experimental allergic encephalomyelitis (CREAE) 13–15 days after the injection of spinal cord homogenate (SCH) in Freund’s complete adjuvant, mice were given five daily injections of phosphate‐buffered saline (PBS) (a) or 100 µg of anti‐CD44 mAb IM7 (b). On day 18 postinjection (p.i.), spinal cords were removed, and sections were made and stained with haematoxylin and eosin. (a) A section from a control mouse with EAE containing large numbers of infiltrating cells. (b) A section from a corresponding area from a clinically well IM7‐treated mouse. Note the lack of infiltrating mononuclear cells.

Techniques Used: Injection, Mouse Assay, Staining

Anti‐CD44 monoclonal antibody (mAb) IM7 inhibits the development and ameliorates the severity of established clinical disease. (a) Mice were injected with mouse spinal cord homogenate (SCH) in Freund’s adjuvant on days 0 and 7 and then injected intraperitoneally (i.p.), daily from day 12 postinjection (p.i.), with 0·1 ml of phosphate‐buffered saline (PBS) (untreated) (•), isotype rat immunoglobulin G2b (IgG2b) (YTS 169) control (▪) or CD44‐specific mAb IM7 (▴). (b) Animals were injected with SCH in Freund’s adjuvant on days 0 and 7. Following the onset of clinical disease on days 13–15 p.i., chronic‐relapsing experimental allergic encephalomyelitis (CREAE) mice were treated (▴) with five daily i.p. injections of 100 µg of anti‐CD44 mAb IM7 (•) or rat immunoglobulin control (▪). For both (a) and (b), the results represent the mean ± SEM clinical score of animals within each group following the start of mAb treatment.
Figure Legend Snippet: Anti‐CD44 monoclonal antibody (mAb) IM7 inhibits the development and ameliorates the severity of established clinical disease. (a) Mice were injected with mouse spinal cord homogenate (SCH) in Freund’s adjuvant on days 0 and 7 and then injected intraperitoneally (i.p.), daily from day 12 postinjection (p.i.), with 0·1 ml of phosphate‐buffered saline (PBS) (untreated) (•), isotype rat immunoglobulin G2b (IgG2b) (YTS 169) control (▪) or CD44‐specific mAb IM7 (▴). (b) Animals were injected with SCH in Freund’s adjuvant on days 0 and 7. Following the onset of clinical disease on days 13–15 p.i., chronic‐relapsing experimental allergic encephalomyelitis (CREAE) mice were treated (▴) with five daily i.p. injections of 100 µg of anti‐CD44 mAb IM7 (•) or rat immunoglobulin control (▪). For both (a) and (b), the results represent the mean ± SEM clinical score of animals within each group following the start of mAb treatment.

Techniques Used: Mouse Assay, Injection

20) Product Images from "mPGES-1-Mediated Production of PGE2 and EP4 Receptor Sensing Regulate T Cell Colonic Inflammation"

Article Title: mPGES-1-Mediated Production of PGE2 and EP4 Receptor Sensing Regulate T Cell Colonic Inflammation

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02954

Paracrine mPGES-1-deficiency in non-lymphoid cells facilitates colitis by inhibiting generation of CD4 + FoxP3 + cells . (A) Weight loss in Rag1 −/− or Rag1 −/− x mPGES-1 −/− mice that received a transfer of WT Teff donor cells. (B) Segregated colon pathology scores from both cohorts. Flow cytometry analysis of (C) mLN and (D) cLP CD4 + populations at the end of the experiment (10 weeks), indicating intracellular expression of RORγt and FoxP3 in summarized results from 3 experiments. (E) Fluorescence microscopy analysis of colon sections denoting CD4 + cell infiltrates. Blue = DAPI, Gray = CD3ε, Green = RORγt, and Red = FoxP3. ** P
Figure Legend Snippet: Paracrine mPGES-1-deficiency in non-lymphoid cells facilitates colitis by inhibiting generation of CD4 + FoxP3 + cells . (A) Weight loss in Rag1 −/− or Rag1 −/− x mPGES-1 −/− mice that received a transfer of WT Teff donor cells. (B) Segregated colon pathology scores from both cohorts. Flow cytometry analysis of (C) mLN and (D) cLP CD4 + populations at the end of the experiment (10 weeks), indicating intracellular expression of RORγt and FoxP3 in summarized results from 3 experiments. (E) Fluorescence microscopy analysis of colon sections denoting CD4 + cell infiltrates. Blue = DAPI, Gray = CD3ε, Green = RORγt, and Red = FoxP3. ** P

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Expressing, Fluorescence, Microscopy

mPGES-1-deficiency in T effector cells protects against colitis. (A) Weight loss in Rag1 −/− mice that received transfer of 1 × 10 6 CD4 + CD25 − CD45RB hi T cells (Teff) from WT or mPGES-1 −/− donors. The dashed lines correspond to mice that received WT Teff and WT or mPGES-1 −/− CD4 + CD25 + (Treg) co-transfers. (B) Colon pathology scores from cohorts receiving transfers of WT or mPGES-1 −/− Teff cells. Flow cytometry analysis of the (C) mesenteric lymph nodes (mLN) and (D) colon lamina propria (cLP) CD4 + populations at the end of the experiment (week 10), with representative dot plots indicating intracellular expression of RORγt and FoxP3 and graphs below indicating summarized results from 4 experiments. ** indicates a significant difference with P
Figure Legend Snippet: mPGES-1-deficiency in T effector cells protects against colitis. (A) Weight loss in Rag1 −/− mice that received transfer of 1 × 10 6 CD4 + CD25 − CD45RB hi T cells (Teff) from WT or mPGES-1 −/− donors. The dashed lines correspond to mice that received WT Teff and WT or mPGES-1 −/− CD4 + CD25 + (Treg) co-transfers. (B) Colon pathology scores from cohorts receiving transfers of WT or mPGES-1 −/− Teff cells. Flow cytometry analysis of the (C) mesenteric lymph nodes (mLN) and (D) colon lamina propria (cLP) CD4 + populations at the end of the experiment (week 10), with representative dot plots indicating intracellular expression of RORγt and FoxP3 and graphs below indicating summarized results from 4 experiments. ** indicates a significant difference with P

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Expressing

Deficiency in CD4-intrinsic mPGES-1 impairs Teff CD4 + cell expansion but enhances Treg localization and RORγt expression in the colonic lamina propria. (A,B) Rag1 −/− recipient mice received a co-transfer of a 1:1 mix of CD45.1 + WT (blue) and CD45.2 + mPGES-1 −/− (red) Teff cells. Flow cytometric analysis of the (A) mLN and (B) cLP CD4 + populations, with representative dot plots indicating intracellular expression of CD45.1 or CD45.2 congenic marker expression together with RORγt. In the cLP plot (B) , the shaded box indicates a unique RORγt hi population of mPGES-1 deficient cells in the cLP. Graphs on the right indicate the proportions and total numbers for each group. (C) Co-transfer of either CD45.1 + WT Treg with CD45.2 + mPGES-1 −/− Teff cells or CD45.2 + mPGES-1 −−/− Treg cells with CD45.1 + WT Teff into Rag1 −/− recipients. Transfers were always performed with a 2:1 Teff:Treg ratio. In the cLP, mPGES-1 −/− CD4 + T cells are able to acquire higher RORγt expression than WT cells (shaded boxes). These CD4 + RORγt hi cells arise from both mPGES-1 −/− Teff cells and mature Treg cells. Graphs on the bottom show the proportions of WT or mPGES-1 −/− Treg cells that are either RORγt − or RORγt + in the mLN or the cLP. ** P
Figure Legend Snippet: Deficiency in CD4-intrinsic mPGES-1 impairs Teff CD4 + cell expansion but enhances Treg localization and RORγt expression in the colonic lamina propria. (A,B) Rag1 −/− recipient mice received a co-transfer of a 1:1 mix of CD45.1 + WT (blue) and CD45.2 + mPGES-1 −/− (red) Teff cells. Flow cytometric analysis of the (A) mLN and (B) cLP CD4 + populations, with representative dot plots indicating intracellular expression of CD45.1 or CD45.2 congenic marker expression together with RORγt. In the cLP plot (B) , the shaded box indicates a unique RORγt hi population of mPGES-1 deficient cells in the cLP. Graphs on the right indicate the proportions and total numbers for each group. (C) Co-transfer of either CD45.1 + WT Treg with CD45.2 + mPGES-1 −/− Teff cells or CD45.2 + mPGES-1 −−/− Treg cells with CD45.1 + WT Teff into Rag1 −/− recipients. Transfers were always performed with a 2:1 Teff:Treg ratio. In the cLP, mPGES-1 −/− CD4 + T cells are able to acquire higher RORγt expression than WT cells (shaded boxes). These CD4 + RORγt hi cells arise from both mPGES-1 −/− Teff cells and mature Treg cells. Graphs on the bottom show the proportions of WT or mPGES-1 −/− Treg cells that are either RORγt − or RORγt + in the mLN or the cLP. ** P

Techniques Used: Expressing, Mouse Assay, Flow Cytometry, Marker

EP4 deficient CD4 + T effector cells have severely blunted colitogenic potential due to impaired proliferative capacity . (A) Weight loss in Rag1 −/− mice that received transfer of Teff cells from EP4 fl/fl (EP4 WT ) or CD4 Cre × EP4 fl/fl (EP4 Δ CD 4 ) donor mice. (B) Colon pathology scores from both cohorts. Flow cytometry analysis of the (C) mLN and (D) cLP CD4 + populations at the end of the experiment (week 10), indicating intracellular expression of RORγt and FoxP3 in summarized results from 4 experiments. (E) Colon images detailing lamina propria T cell infiltrates in both groups, with magnified inserts on the right-hand side. Blue = DAPI, Gray = CD3ε, Green = RORγt, and Red = FoxP3. ** P
Figure Legend Snippet: EP4 deficient CD4 + T effector cells have severely blunted colitogenic potential due to impaired proliferative capacity . (A) Weight loss in Rag1 −/− mice that received transfer of Teff cells from EP4 fl/fl (EP4 WT ) or CD4 Cre × EP4 fl/fl (EP4 Δ CD 4 ) donor mice. (B) Colon pathology scores from both cohorts. Flow cytometry analysis of the (C) mLN and (D) cLP CD4 + populations at the end of the experiment (week 10), indicating intracellular expression of RORγt and FoxP3 in summarized results from 4 experiments. (E) Colon images detailing lamina propria T cell infiltrates in both groups, with magnified inserts on the right-hand side. Blue = DAPI, Gray = CD3ε, Green = RORγt, and Red = FoxP3. ** P

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Expressing

PGE 2 controls phosphorylation of STAT3 in colon-infiltrating T cells. Individual colons from the 3 different groups of colitic mice indicated (1 = WT Teff → Rag1 −/− , 2 = EP4 Δ CD 4 Teff → Rag1 −/− , 3 = WT Teff → Rag1 −/− x mPGES-1 −/− ) were processed and stained for fluorescence microscopy evaluation DAPI, CD3ε, RORγt, FoxP3, and pSTAT3. (A) Quantification of total CD3ε + and CD3ε − cells in 15 regions/colon containing a total of 15–30K cells. (B) Quantification of total CD3ε + FoxP3 + and CD3ε + RORγt + cells in the same groups. (C) Representative colon sections depicting co-localization of RORγt (green), pSTAT3 (red) and CD3 (gray) in the upper row, or of FoxP3 (green), pSTAT3 (red) with CD3 (gray) in the lower row in the same 3 groups. (D) Summary of the quantification of pSTAT3 + cells within the indicated CD3ε + subsets (single RORγt + , RORγt − FoxP3 − or single FoxP3 + ). * P
Figure Legend Snippet: PGE 2 controls phosphorylation of STAT3 in colon-infiltrating T cells. Individual colons from the 3 different groups of colitic mice indicated (1 = WT Teff → Rag1 −/− , 2 = EP4 Δ CD 4 Teff → Rag1 −/− , 3 = WT Teff → Rag1 −/− x mPGES-1 −/− ) were processed and stained for fluorescence microscopy evaluation DAPI, CD3ε, RORγt, FoxP3, and pSTAT3. (A) Quantification of total CD3ε + and CD3ε − cells in 15 regions/colon containing a total of 15–30K cells. (B) Quantification of total CD3ε + FoxP3 + and CD3ε + RORγt + cells in the same groups. (C) Representative colon sections depicting co-localization of RORγt (green), pSTAT3 (red) and CD3 (gray) in the upper row, or of FoxP3 (green), pSTAT3 (red) with CD3 (gray) in the lower row in the same 3 groups. (D) Summary of the quantification of pSTAT3 + cells within the indicated CD3ε + subsets (single RORγt + , RORγt − FoxP3 − or single FoxP3 + ). * P

Techniques Used: Mouse Assay, Staining, Fluorescence, Microscopy

21) Product Images from "Differential Requirement for the CD45 Splicing Regulator hnRNPLL for Accumulation of NKT and Conventional T Cells"

Article Title: Differential Requirement for the CD45 Splicing Regulator hnRNPLL for Accumulation of NKT and Conventional T Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0026440

hnRNPLL is required for the splicing of CD45 isoforms in NKT cells. Representative overlay histograms of (A) CD45 and (B) CD45RA, CD45RB and CD45RC expressions on wild type (+/+) (shaded grey) and Hnrpll thu/thu (thu/thu) (black line) CD4 T cells and NKT cells in the thymus (Data are representative of two independent experiments with n = 2–3 mice per group in each).
Figure Legend Snippet: hnRNPLL is required for the splicing of CD45 isoforms in NKT cells. Representative overlay histograms of (A) CD45 and (B) CD45RA, CD45RB and CD45RC expressions on wild type (+/+) (shaded grey) and Hnrpll thu/thu (thu/thu) (black line) CD4 T cells and NKT cells in the thymus (Data are representative of two independent experiments with n = 2–3 mice per group in each).

Techniques Used: Mouse Assay

22) Product Images from "The survival of memory CD8 T cells that is mediated by IL-15 correlates with sustained protection against malaria 5The survival of memory CD8 T cells that is mediated by IL-15 correlates with sustained protection against malaria 5 6"

Article Title: The survival of memory CD8 T cells that is mediated by IL-15 correlates with sustained protection against malaria 5The survival of memory CD8 T cells that is mediated by IL-15 correlates with sustained protection against malaria 5 6

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

doi: 10.4049/jimmunol.1203396

IL-15KO CD8 T cells respond to antigen from Pb γ-spz Wt and IL-15KO mice (3–5 mice/group) were immunized with 3 weekly doses of Pb γ-spz followed one week later by infectious sporozoite challenge as described in materials and methods. (A) Livers from naïve, immune, or immune/challenged (CH) mice were isolated 1 week after last exposure to antigen and stained with anti-CD3, -CD8, -CD44, -CD62L mAbs to determine the number of total CD8 T cells, CD8 T E/EM cells and CD8 T CM cells. The results show the number of cells as the mean ± SD (B) IHMC were isolated from wt and IL-15KO mice one week following the 3 rd immunization with Pb γ-spz. IFN-γ was detected by cytokine secretion assay, as described in Materials and Methods. CD8 T cells were enriched by magnetic bead separation and CD44 hi CD45RB hi or CD44 hi CD45RB lo T cells were identified by flow cytometry. The results are representative of several experiments performed under the same conditions and show the number of IFN-γ secreting CD8 T CM cells and CD8 T E/EM cells per 10 6 IHMC determined for each individual mouse and are presented as the mean ± SD, *p
Figure Legend Snippet: IL-15KO CD8 T cells respond to antigen from Pb γ-spz Wt and IL-15KO mice (3–5 mice/group) were immunized with 3 weekly doses of Pb γ-spz followed one week later by infectious sporozoite challenge as described in materials and methods. (A) Livers from naïve, immune, or immune/challenged (CH) mice were isolated 1 week after last exposure to antigen and stained with anti-CD3, -CD8, -CD44, -CD62L mAbs to determine the number of total CD8 T cells, CD8 T E/EM cells and CD8 T CM cells. The results show the number of cells as the mean ± SD (B) IHMC were isolated from wt and IL-15KO mice one week following the 3 rd immunization with Pb γ-spz. IFN-γ was detected by cytokine secretion assay, as described in Materials and Methods. CD8 T cells were enriched by magnetic bead separation and CD44 hi CD45RB hi or CD44 hi CD45RB lo T cells were identified by flow cytometry. The results are representative of several experiments performed under the same conditions and show the number of IFN-γ secreting CD8 T CM cells and CD8 T E/EM cells per 10 6 IHMC determined for each individual mouse and are presented as the mean ± SD, *p

Techniques Used: Mouse Assay, Isolation, Staining, Flow Cytometry, Cytometry

23) Product Images from "Rapid Acquisition of Tissue-specific Homing Phenotypes by CD4+ T Cells Activated in Cutaneous or Mucosal Lymphoid Tissues"

Article Title: Rapid Acquisition of Tissue-specific Homing Phenotypes by CD4+ T Cells Activated in Cutaneous or Mucosal Lymphoid Tissues

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20011502

CD4 + T cells activated in PLNs and MLNs differentially upregulate P-lig and α4β7. (A) Percentage of OVA-specific KJ1–26 + cells among gated CD4 + T cells isolated from peripheral blood at the indicated times after intraperitoneal injection of OVA plus LPS. Data are mean and SD of values obtained from five mice at each time point. (B) Representative flow cytometry data of cellular CFSE content and KJ1–26 staining on gated CD4 + T cells isolated from the indicated tissues 2 d after intraperitoneal immunization of DO11.10 adoptive transfer recipients with OVA plus LPS. The horizontal marker in the histograms represents the CFSE fluorescence intensity of naive CD4 + KJ1–26 + T cells isolated from animals immunized with LPS alone (data not shown). (C) Expression of α4β7 and P-lig by gated CD4 + KJ1–26 + cells isolated from PLNs (• and ○) and MLNs (▪ and □) 2 d after intraperitoneal immunization of DO11.10 adoptive transfer recipients with OVA plus LPS (black symbols) or LPS alone (white symbols). Each data point represents a measurement from an individual animal. (D) Representative flow cytometry data of α4β7 and P-lig staining on gated CD4 + KJ1–26 + cells isolated from PLNs (top) and MLN (bottom) 2 d after immunization of DO11.10 adoptive transfer recipients with OVA plus LPS (left) or LPS alone (right). The quadrant gate used to define the P-lig + and α4β7 hi populations in C is indicated. (E) Mean fluorescence intensity (MFI) of α4β7 (left) and P-lig (right) staining on gated CD4 + KJ1–26 + cells isolated from the MLNs (▪) or PLNs (•) 2 d after intraperitoneal immunization of DO11.10 adoptive transfer recipients with OVA plus LPS as a function of cell division (as determined by CFSE content). Data are mean and SE of values obtained from four (α4β7) or five (P-lig) mice. N represents the MFI of α4β7 or P-lig staining on naive cells isolated from animals immunized with LPS alone. Dotted lines represent background MFI of cells stained with an isotype control (left) or unstained cells (right).
Figure Legend Snippet: CD4 + T cells activated in PLNs and MLNs differentially upregulate P-lig and α4β7. (A) Percentage of OVA-specific KJ1–26 + cells among gated CD4 + T cells isolated from peripheral blood at the indicated times after intraperitoneal injection of OVA plus LPS. Data are mean and SD of values obtained from five mice at each time point. (B) Representative flow cytometry data of cellular CFSE content and KJ1–26 staining on gated CD4 + T cells isolated from the indicated tissues 2 d after intraperitoneal immunization of DO11.10 adoptive transfer recipients with OVA plus LPS. The horizontal marker in the histograms represents the CFSE fluorescence intensity of naive CD4 + KJ1–26 + T cells isolated from animals immunized with LPS alone (data not shown). (C) Expression of α4β7 and P-lig by gated CD4 + KJ1–26 + cells isolated from PLNs (• and ○) and MLNs (▪ and □) 2 d after intraperitoneal immunization of DO11.10 adoptive transfer recipients with OVA plus LPS (black symbols) or LPS alone (white symbols). Each data point represents a measurement from an individual animal. (D) Representative flow cytometry data of α4β7 and P-lig staining on gated CD4 + KJ1–26 + cells isolated from PLNs (top) and MLN (bottom) 2 d after immunization of DO11.10 adoptive transfer recipients with OVA plus LPS (left) or LPS alone (right). The quadrant gate used to define the P-lig + and α4β7 hi populations in C is indicated. (E) Mean fluorescence intensity (MFI) of α4β7 (left) and P-lig (right) staining on gated CD4 + KJ1–26 + cells isolated from the MLNs (▪) or PLNs (•) 2 d after intraperitoneal immunization of DO11.10 adoptive transfer recipients with OVA plus LPS as a function of cell division (as determined by CFSE content). Data are mean and SE of values obtained from four (α4β7) or five (P-lig) mice. N represents the MFI of α4β7 or P-lig staining on naive cells isolated from animals immunized with LPS alone. Dotted lines represent background MFI of cells stained with an isotype control (left) or unstained cells (right).

Techniques Used: Isolation, Injection, Mouse Assay, Flow Cytometry, Cytometry, Staining, Adoptive Transfer Assay, Marker, Fluorescence, Expressing

α4β7 and P-lig define three CD4 + memory T cell populations that differentially localize in intestinal and cutaneous lymphoid organs. (Left) CD4 and CD45RB expression by lymphocytes isolated from the cutaneous PLNs of a > 1-y-old BALB/c mouse. The gates used to define ‘naive’ (CD4 + CD45RB + ) and ‘memory’ (CD4 + CD45RB − ) T cells are shown. (Right) α4β7 and P-lig expression by gated naive and memory CD4 + T cells isolated from the indicated lymphoid tissues of a > 1-y-old BALB/c mouse.
Figure Legend Snippet: α4β7 and P-lig define three CD4 + memory T cell populations that differentially localize in intestinal and cutaneous lymphoid organs. (Left) CD4 and CD45RB expression by lymphocytes isolated from the cutaneous PLNs of a > 1-y-old BALB/c mouse. The gates used to define ‘naive’ (CD4 + CD45RB + ) and ‘memory’ (CD4 + CD45RB − ) T cells are shown. (Right) α4β7 and P-lig expression by gated naive and memory CD4 + T cells isolated from the indicated lymphoid tissues of a > 1-y-old BALB/c mouse.

Techniques Used: Expressing, Isolation

24) Product Images from "Hsp65-Producing Lactococcus lactis Prevents Inflammatory Intestinal Disease in Mice by IL-10- and TLR2-Dependent Pathways"

Article Title: Hsp65-Producing Lactococcus lactis Prevents Inflammatory Intestinal Disease in Mice by IL-10- and TLR2-Dependent Pathways

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00030

Hsp65-producing L. lactis (Hsp65-LL) induces increased frequencies of regulatory T cells (Tregs) in mice . C57BL/6 mice were pretreated or not (naïve) with medium (CT), empty vector-harboring L. lactis (CT-LL), or Hsp65-LL for 4 days and colitis was induced 10 days later by 1.5% DSS in drinking water. Seventy-two hours after DSS administration, mice were sacrificed and mesenteric lymph nodes (mLNs) removed. Cells were stained with fluorescein isothiocyanate-conjugated (FITC)-anti-CD4, PE-anti-Foxp3, Bio-CD25 and CY5.5-STV (A) or with FITC-anti-CD4, PE-anti-CD25, Bio-LAP, and Cy5.5-STV (B) . Alternatively, C57BL/6 mice were orally pretreated or not (naïve) with Hsp65-LL during 4 days. Either 4 or 10 days after the last day of oral treatment frequencies of Treg populations were analyzed by flow cytometry. (C) Spleen and (D) mLN cells were stained with Cy5.5 anti-CD4 and PE anti-Foxp3. (E) Spleen cells were stained with Cy5.5 anti-CD4; biotin anti-LAP, and APC streptavidin. CD4 + Foxp3 + and CD4 + LAP + cells were gated on lymphocyte population ( N = 4). Results are representative of three independent experiments. Bar graphs are shown as mean ± SEM. Analysis of variance, Tukey’s post hoc test, p
Figure Legend Snippet: Hsp65-producing L. lactis (Hsp65-LL) induces increased frequencies of regulatory T cells (Tregs) in mice . C57BL/6 mice were pretreated or not (naïve) with medium (CT), empty vector-harboring L. lactis (CT-LL), or Hsp65-LL for 4 days and colitis was induced 10 days later by 1.5% DSS in drinking water. Seventy-two hours after DSS administration, mice were sacrificed and mesenteric lymph nodes (mLNs) removed. Cells were stained with fluorescein isothiocyanate-conjugated (FITC)-anti-CD4, PE-anti-Foxp3, Bio-CD25 and CY5.5-STV (A) or with FITC-anti-CD4, PE-anti-CD25, Bio-LAP, and Cy5.5-STV (B) . Alternatively, C57BL/6 mice were orally pretreated or not (naïve) with Hsp65-LL during 4 days. Either 4 or 10 days after the last day of oral treatment frequencies of Treg populations were analyzed by flow cytometry. (C) Spleen and (D) mLN cells were stained with Cy5.5 anti-CD4 and PE anti-Foxp3. (E) Spleen cells were stained with Cy5.5 anti-CD4; biotin anti-LAP, and APC streptavidin. CD4 + Foxp3 + and CD4 + LAP + cells were gated on lymphocyte population ( N = 4). Results are representative of three independent experiments. Bar graphs are shown as mean ± SEM. Analysis of variance, Tukey’s post hoc test, p

Techniques Used: Mouse Assay, Plasmid Preparation, Staining, Flow Cytometry, Cytometry

25) Product Images from "The survival of memory CD8 T cells that is mediated by IL-15 correlates with sustained protection against malaria 5The survival of memory CD8 T cells that is mediated by IL-15 correlates with sustained protection against malaria 5 6"

Article Title: The survival of memory CD8 T cells that is mediated by IL-15 correlates with sustained protection against malaria 5The survival of memory CD8 T cells that is mediated by IL-15 correlates with sustained protection against malaria 5 6

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

doi: 10.4049/jimmunol.1203396

IL-15KO CD8 T cells respond to antigen from Pb γ-spz Wt and IL-15KO mice (3–5 mice/group) were immunized with 3 weekly doses of Pb γ-spz followed one week later by infectious sporozoite challenge as described in materials and methods. (A) Livers from naïve, immune, or immune/challenged (CH) mice were isolated 1 week after last exposure to antigen and stained with anti-CD3, -CD8, -CD44, -CD62L mAbs to determine the number of total CD8 T cells, CD8 T E/EM cells and CD8 T CM cells. The results show the number of cells as the mean ± SD (B) IHMC were isolated from wt and IL-15KO mice one week following the 3 rd immunization with Pb γ-spz. IFN-γ was detected by cytokine secretion assay, as described in Materials and Methods. CD8 T cells were enriched by magnetic bead separation and CD44 hi CD45RB hi or CD44 hi CD45RB lo T cells were identified by flow cytometry. The results are representative of several experiments performed under the same conditions and show the number of IFN-γ secreting CD8 T CM cells and CD8 T E/EM cells per 10 6 IHMC determined for each individual mouse and are presented as the mean ± SD, *p
Figure Legend Snippet: IL-15KO CD8 T cells respond to antigen from Pb γ-spz Wt and IL-15KO mice (3–5 mice/group) were immunized with 3 weekly doses of Pb γ-spz followed one week later by infectious sporozoite challenge as described in materials and methods. (A) Livers from naïve, immune, or immune/challenged (CH) mice were isolated 1 week after last exposure to antigen and stained with anti-CD3, -CD8, -CD44, -CD62L mAbs to determine the number of total CD8 T cells, CD8 T E/EM cells and CD8 T CM cells. The results show the number of cells as the mean ± SD (B) IHMC were isolated from wt and IL-15KO mice one week following the 3 rd immunization with Pb γ-spz. IFN-γ was detected by cytokine secretion assay, as described in Materials and Methods. CD8 T cells were enriched by magnetic bead separation and CD44 hi CD45RB hi or CD44 hi CD45RB lo T cells were identified by flow cytometry. The results are representative of several experiments performed under the same conditions and show the number of IFN-γ secreting CD8 T CM cells and CD8 T E/EM cells per 10 6 IHMC determined for each individual mouse and are presented as the mean ± SD, *p

Techniques Used: Mouse Assay, Isolation, Staining, Flow Cytometry, Cytometry

CD8 T CM cells, having elevated expression of IL-15Rβ (CD122), are the primary cells responding to IL-15 IHMC were isolated from C57BL/6 mice (3 per group) 1 month after a tertiary immunization with Pb γ-spz. (A) CD62L + and CD62L − T cells were separated by magnetic bead isolation procedure; a second round of isolation using CD8 magnetic beads resulted in the following subpopulations: CD8 + CD62L + and CD8 + CD62L − T cells as well as CD8 − CD62L + and CD8 − CD62L − T cells. Each T cell subset was cultured at 4 × 10 5 cells/0.2 ml culture medium for 96 hours in the presence of the indicted concentrations of IL-15; during the last 16hrs of culture, 1μCi of 3 H-TdR was added to each well. Results are presented as the mean CPM ± SD of 3 H-TdR uptake in triplicate wells and are representative of 3 separate experiments. (B) Liver CD8 T cells, isolated by negative magnetic beads selection, were stained with CFSE and incubated for 7 days in the presence of 100 ng/ml IL-15, or 100ng/ml IL-2. Harvested cells were stained with mAbs against CD8, CD44 and CD45RB. Lymphocytes were gated on a forward-side scatter plot, and gates were applied to identify CD8 + T cells that expressed either CD44 hi CD45RB hi (T CM cells) or CD44 hi CD45RB lo (T E/EM cells) phenotypes. Histogram plots represent CFSE dilution with gray shaded area representing CD8 T CM cells and solid contour line representing CD8 T E/EM cells. These results represent one of two separate experiments. (C) Liver CD8 T cells were cultured in the presence of either 100 ng/ml IL-15, or medium alone. Cells were harvested on day 1, 3, and 7 and stained with mAbs against CD44 and CD45RB as described above, fixed, permeabilized and stained with mAbs against Bcl-2. Mean fluorescent intensity (determined for each individual mouse and is presented as the mean ± SD) of Bcl-2 expression of T CM or T E/EM . Bcl-2 determinations on CD8 T CM and CD8 T E/EM cells prior to culture showed MFI=189± 45 for CD8 T CM and MFI= 76± 35 for CD8 T E/EM . Results are representative of 3 separate experiments. *p
Figure Legend Snippet: CD8 T CM cells, having elevated expression of IL-15Rβ (CD122), are the primary cells responding to IL-15 IHMC were isolated from C57BL/6 mice (3 per group) 1 month after a tertiary immunization with Pb γ-spz. (A) CD62L + and CD62L − T cells were separated by magnetic bead isolation procedure; a second round of isolation using CD8 magnetic beads resulted in the following subpopulations: CD8 + CD62L + and CD8 + CD62L − T cells as well as CD8 − CD62L + and CD8 − CD62L − T cells. Each T cell subset was cultured at 4 × 10 5 cells/0.2 ml culture medium for 96 hours in the presence of the indicted concentrations of IL-15; during the last 16hrs of culture, 1μCi of 3 H-TdR was added to each well. Results are presented as the mean CPM ± SD of 3 H-TdR uptake in triplicate wells and are representative of 3 separate experiments. (B) Liver CD8 T cells, isolated by negative magnetic beads selection, were stained with CFSE and incubated for 7 days in the presence of 100 ng/ml IL-15, or 100ng/ml IL-2. Harvested cells were stained with mAbs against CD8, CD44 and CD45RB. Lymphocytes were gated on a forward-side scatter plot, and gates were applied to identify CD8 + T cells that expressed either CD44 hi CD45RB hi (T CM cells) or CD44 hi CD45RB lo (T E/EM cells) phenotypes. Histogram plots represent CFSE dilution with gray shaded area representing CD8 T CM cells and solid contour line representing CD8 T E/EM cells. These results represent one of two separate experiments. (C) Liver CD8 T cells were cultured in the presence of either 100 ng/ml IL-15, or medium alone. Cells were harvested on day 1, 3, and 7 and stained with mAbs against CD44 and CD45RB as described above, fixed, permeabilized and stained with mAbs against Bcl-2. Mean fluorescent intensity (determined for each individual mouse and is presented as the mean ± SD) of Bcl-2 expression of T CM or T E/EM . Bcl-2 determinations on CD8 T CM and CD8 T E/EM cells prior to culture showed MFI=189± 45 for CD8 T CM and MFI= 76± 35 for CD8 T E/EM . Results are representative of 3 separate experiments. *p

Techniques Used: Expressing, Isolation, Mouse Assay, Magnetic Beads, Cell Culture, Selection, Staining, Incubation

26) Product Images from "Differential Requirement for the CD45 Splicing Regulator hnRNPLL for Accumulation of NKT and Conventional T Cells"

Article Title: Differential Requirement for the CD45 Splicing Regulator hnRNPLL for Accumulation of NKT and Conventional T Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0026440

Normal contribution of Hnrpll thu/thu NKT cells, but a cell autonomous decrease in NK1.1 in the mixed bone marrow chimeras. Irradiated C57BL/6 mice were reconstituted with an equal mixture of CD45.1-marked wild-type bone marrow and CD45.2-marked Hnrpll thu/thu bone marrow cells. After hemopoietic reconstitution, recipient mice were analysed for B, NKT and conventional T cell populations. (A) Graph shows the ratio of CD45.2 + wild type and Hnrpll thu/thu derived T cells to CD45.2 + wild type and Hnrpll thu/thu derived B cells in the spleen of chimeric recipient mice. (B) Graph shows the ratio of CD45.2 + wild type and Hnrpll thu/thu derived NKT cells to CD45.2 + wild type and Hnrpll thu/thu derived B cells in the spleen of chimeric recipient mice. (C, D) Representative overlay histograms comparing the expression of NK1.1 on wild type (shaded grey) and Hnrpll thu/thu (black line) derived NKT cells in the (b) thymus and (c) spleen of individual recipient mice from mixed bone marrow chimeras. Graphs show the geometric mean florescence intensity (MFI) of NK1.1 in the CD45.2 + wild type or Hnrpll thu/thu derived NKT cells in the (b) thymus and (c) spleen of individual recipient mice from mixed bone marrow chimeras. (n = 6 for wild type and n = 5 for Hnrpll thu/thu ). Bars represent the mean ± s.d. values.
Figure Legend Snippet: Normal contribution of Hnrpll thu/thu NKT cells, but a cell autonomous decrease in NK1.1 in the mixed bone marrow chimeras. Irradiated C57BL/6 mice were reconstituted with an equal mixture of CD45.1-marked wild-type bone marrow and CD45.2-marked Hnrpll thu/thu bone marrow cells. After hemopoietic reconstitution, recipient mice were analysed for B, NKT and conventional T cell populations. (A) Graph shows the ratio of CD45.2 + wild type and Hnrpll thu/thu derived T cells to CD45.2 + wild type and Hnrpll thu/thu derived B cells in the spleen of chimeric recipient mice. (B) Graph shows the ratio of CD45.2 + wild type and Hnrpll thu/thu derived NKT cells to CD45.2 + wild type and Hnrpll thu/thu derived B cells in the spleen of chimeric recipient mice. (C, D) Representative overlay histograms comparing the expression of NK1.1 on wild type (shaded grey) and Hnrpll thu/thu (black line) derived NKT cells in the (b) thymus and (c) spleen of individual recipient mice from mixed bone marrow chimeras. Graphs show the geometric mean florescence intensity (MFI) of NK1.1 in the CD45.2 + wild type or Hnrpll thu/thu derived NKT cells in the (b) thymus and (c) spleen of individual recipient mice from mixed bone marrow chimeras. (n = 6 for wild type and n = 5 for Hnrpll thu/thu ). Bars represent the mean ± s.d. values.

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Expressing

27) Product Images from "Expanded B cell population blocks regulatory T cells and exacerbates ileitis in a murine model of Crohn disease"

Article Title: Expanded B cell population blocks regulatory T cells and exacerbates ileitis in a murine model of Crohn disease

Journal: Journal of Clinical Investigation

doi: 10.1172/JCI200420855

SAMP1/YitFc MLN αEβ7 + CD4 + T cells possess a regulatory phenotype. ( A ) Comparison of the percentage of MLN cells, determined by flow cytometry, that express high levels of CD69 ( n = 9 mice), CD25 ( n = 9), L-selectin ( n = 14), and CD45RB ( n = 8) within the αEβ7 + CD4 + and αEβ7 – CD4 + T cell subsets. ( B ) Levels of secreted TNF-α, IL-2, and IL-10, measured by cytometric bead array in triplicate, from 48-hour cultures of SAMP1/YitFc MLN unfractionated CD4 + T cells (Total CD4 + ), αE + CD4 + T cells, or αE – CD4 + T cells (105 cells/well), stimulated with immobilized anti-CD3 and soluble anti-CD28. Values represent the averages of three independent experiments for each group. *Significantly different ( P
Figure Legend Snippet: SAMP1/YitFc MLN αEβ7 + CD4 + T cells possess a regulatory phenotype. ( A ) Comparison of the percentage of MLN cells, determined by flow cytometry, that express high levels of CD69 ( n = 9 mice), CD25 ( n = 9), L-selectin ( n = 14), and CD45RB ( n = 8) within the αEβ7 + CD4 + and αEβ7 – CD4 + T cell subsets. ( B ) Levels of secreted TNF-α, IL-2, and IL-10, measured by cytometric bead array in triplicate, from 48-hour cultures of SAMP1/YitFc MLN unfractionated CD4 + T cells (Total CD4 + ), αE + CD4 + T cells, or αE – CD4 + T cells (105 cells/well), stimulated with immobilized anti-CD3 and soluble anti-CD28. Values represent the averages of three independent experiments for each group. *Significantly different ( P

Techniques Used: Flow Cytometry, Cytometry, Mouse Assay

28) Product Images from "Expanded B cell population blocks regulatory T cells and exacerbates ileitis in a murine model of Crohn disease"

Article Title: Expanded B cell population blocks regulatory T cells and exacerbates ileitis in a murine model of Crohn disease

Journal: Journal of Clinical Investigation

doi: 10.1172/JCI200420855

SAMP1/YitFc MLN αEβ7 + CD4 + T cells possess a regulatory phenotype. ( A ) Comparison of the percentage of MLN cells, determined by flow cytometry, that express high levels of CD69 ( n = 9 mice), CD25 ( n = 9), L-selectin ( n = 14), and CD45RB ( n = 8) within the αEβ7 + CD4 + and αEβ7 – CD4 + T cell subsets. ( B ) Levels of secreted TNF-α, IL-2, and IL-10, measured by cytometric bead array in triplicate, from 48-hour cultures of SAMP1/YitFc MLN unfractionated CD4 + T cells (Total CD4 + ), αE + CD4 + T cells, or αE – CD4 + T cells (105 cells/well), stimulated with immobilized anti-CD3 and soluble anti-CD28. Values represent the averages of three independent experiments for each group. *Significantly different ( P
Figure Legend Snippet: SAMP1/YitFc MLN αEβ7 + CD4 + T cells possess a regulatory phenotype. ( A ) Comparison of the percentage of MLN cells, determined by flow cytometry, that express high levels of CD69 ( n = 9 mice), CD25 ( n = 9), L-selectin ( n = 14), and CD45RB ( n = 8) within the αEβ7 + CD4 + and αEβ7 – CD4 + T cell subsets. ( B ) Levels of secreted TNF-α, IL-2, and IL-10, measured by cytometric bead array in triplicate, from 48-hour cultures of SAMP1/YitFc MLN unfractionated CD4 + T cells (Total CD4 + ), αE + CD4 + T cells, or αE – CD4 + T cells (105 cells/well), stimulated with immobilized anti-CD3 and soluble anti-CD28. Values represent the averages of three independent experiments for each group. *Significantly different ( P

Techniques Used: Flow Cytometry, Cytometry, Mouse Assay

Expanded B cell and α E β 7 + CD4 + T cell populations in SAMP1/YitFc versus AKR MLN. ( A ) Total lymphocyte numbers (mean ± SEM) presented as the percentage of total cells in AKR and SAMP1/YitFc MLNs — as determined by lymphocyte-gated flow cytometry — that are CD4 + T cells ( n = 16 and 32, respectively), CD8 + T cells ( n = 10 and 19, respectively), and B cells ( n = 13 and 24, respectively). Fold increases in the overall size of each of these populations in SAMP1/YitFc versus AKR are indicated. ( B ) Top, CD4 + T cell–gated histograms of β 7 integrin chain expression on MLN cells from SAMP1/YitFc and AKR mice, showing that SAMP1/YitFc mice have an increased percentage of CD4 + T cells expressing high levels of β 7 . Bottom, the β 7 hi cells express β 7 as a dimer with the α E integrin chain, as α E + cells display a 1:1 correlation of αE to β7 expression in β7 versus αE dot plots that is not seen in isotype controls. ( C ) Comparison of the percentage (mean ± SEM) of MLN CD4 + T cells that are αE + in SAMP1/YitFc mice ( n = 31) versus AKR mice ( n = 21). *Significantly greater ( P
Figure Legend Snippet: Expanded B cell and α E β 7 + CD4 + T cell populations in SAMP1/YitFc versus AKR MLN. ( A ) Total lymphocyte numbers (mean ± SEM) presented as the percentage of total cells in AKR and SAMP1/YitFc MLNs — as determined by lymphocyte-gated flow cytometry — that are CD4 + T cells ( n = 16 and 32, respectively), CD8 + T cells ( n = 10 and 19, respectively), and B cells ( n = 13 and 24, respectively). Fold increases in the overall size of each of these populations in SAMP1/YitFc versus AKR are indicated. ( B ) Top, CD4 + T cell–gated histograms of β 7 integrin chain expression on MLN cells from SAMP1/YitFc and AKR mice, showing that SAMP1/YitFc mice have an increased percentage of CD4 + T cells expressing high levels of β 7 . Bottom, the β 7 hi cells express β 7 as a dimer with the α E integrin chain, as α E + cells display a 1:1 correlation of αE to β7 expression in β7 versus αE dot plots that is not seen in isotype controls. ( C ) Comparison of the percentage (mean ± SEM) of MLN CD4 + T cells that are αE + in SAMP1/YitFc mice ( n = 31) versus AKR mice ( n = 21). *Significantly greater ( P

Techniques Used: Flow Cytometry, Cytometry, Expressing, Mouse Assay

29) Product Images from "Bacteria-triggered CD4+ T Regulatory Cells Suppress Helicobacter hepaticus-induced Colitis"

Article Title: Bacteria-triggered CD4+ T Regulatory Cells Suppress Helicobacter hepaticus-induced Colitis

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20020556

CD45RB low CD4 + cells from infected WT mice inhibit IFN-γ production by IL-10 KO CD4 + cells and produce IL-10 after in vitro stimulation with SHelAg. (A) Responder CD4 + cells from infected IL-10 KO mice were cultured with APC, SHelAg, and indicated numbers of CD45RB low CD4 + cells from naive (○) or infected (•) WT mice. IFN-γ was measured in 72-h supernatants. (B) CD45RB low CD4 + cells from naive (□) or infected (▪) WT mice were cultured with IL-10–deficient APC and indicated Ag. IL-10 was measured in 72-h supernatants. No IFN-γ, IL-4, or IL-5 was detected in cultures stimulated with SHelAg plus IL-2. (C) CD45RB low cells from naive or infected WT mice were cultured with IL-10–deficient APC and SHelAg plus IL-2. After removal of supernatants at 72 h, IL-2 was added for an additional 18 h before cells were stimulated with PMA plus ionomycin and analyzed for the expression of IL-10 and IFN-γ by intracellular staining. The FACS ® dot plots shown are gated on CD4 + cells. In the same assay, the cells were negative for intracellular IL-4. The results shown in A–C are representative of two or three experiments performed.
Figure Legend Snippet: CD45RB low CD4 + cells from infected WT mice inhibit IFN-γ production by IL-10 KO CD4 + cells and produce IL-10 after in vitro stimulation with SHelAg. (A) Responder CD4 + cells from infected IL-10 KO mice were cultured with APC, SHelAg, and indicated numbers of CD45RB low CD4 + cells from naive (○) or infected (•) WT mice. IFN-γ was measured in 72-h supernatants. (B) CD45RB low CD4 + cells from naive (□) or infected (▪) WT mice were cultured with IL-10–deficient APC and indicated Ag. IL-10 was measured in 72-h supernatants. No IFN-γ, IL-4, or IL-5 was detected in cultures stimulated with SHelAg plus IL-2. (C) CD45RB low cells from naive or infected WT mice were cultured with IL-10–deficient APC and SHelAg plus IL-2. After removal of supernatants at 72 h, IL-2 was added for an additional 18 h before cells were stimulated with PMA plus ionomycin and analyzed for the expression of IL-10 and IFN-γ by intracellular staining. The FACS ® dot plots shown are gated on CD4 + cells. In the same assay, the cells were negative for intracellular IL-4. The results shown in A–C are representative of two or three experiments performed.

Techniques Used: Infection, Mouse Assay, In Vitro, Cell Culture, Expressing, Staining, FACS

CD45RB low CD4 + cells from H. hepaticus –infected WT mice protect RAG KO animals from colitis induced by IL-10 KO CD4 + cells plus bacterial infection. (A) H. hepaticus –infected RAG KO mice (solid bars) were inoculated with CD4 + cells from infected IL-10 KO mice and CD4 + cells from naive or infected WT mice as indicated (4 × 10 5 of each population). Intestinal pathology was analyzed 4 wk later. Naive (open bar) and infected RAG KO animals receiving no cells were included as controls. Bars represent mean histology scores ± SD of three mice per group except for groups receiving WT cells alone, in which case, due to limited cell numbers, only two mice per group were used. Similar results were seen in ascending colon (although histology scores were lower) and when disease was induced by the transfer of CD4 + cells from naive IL-10 KO mice (not depicted). (B) Infected RAG KO mice were given either no cells or CD4 + cells from infected IL-10 KO mice and CD4 + , CD45RB hi CD4 + , or CD45RB low CD4 + cells from infected WT mice as indicated (4 × 10 5 of each population). Pathology in the cecum (solid bars) and ascending colon (gray bars) were analyzed 4 wk later. Bars represent mean histology scores ± SD of six or seven mice per group pooled from two separate experiments except for the group receiving IL-10 KO CD4 + cells plus infected WT CD4 + cells ( n = 3), which was included in only one of the experiments. Statistical significance was tested for groups receiving IL-10 KO cells. *, P
Figure Legend Snippet: CD45RB low CD4 + cells from H. hepaticus –infected WT mice protect RAG KO animals from colitis induced by IL-10 KO CD4 + cells plus bacterial infection. (A) H. hepaticus –infected RAG KO mice (solid bars) were inoculated with CD4 + cells from infected IL-10 KO mice and CD4 + cells from naive or infected WT mice as indicated (4 × 10 5 of each population). Intestinal pathology was analyzed 4 wk later. Naive (open bar) and infected RAG KO animals receiving no cells were included as controls. Bars represent mean histology scores ± SD of three mice per group except for groups receiving WT cells alone, in which case, due to limited cell numbers, only two mice per group were used. Similar results were seen in ascending colon (although histology scores were lower) and when disease was induced by the transfer of CD4 + cells from naive IL-10 KO mice (not depicted). (B) Infected RAG KO mice were given either no cells or CD4 + cells from infected IL-10 KO mice and CD4 + , CD45RB hi CD4 + , or CD45RB low CD4 + cells from infected WT mice as indicated (4 × 10 5 of each population). Pathology in the cecum (solid bars) and ascending colon (gray bars) were analyzed 4 wk later. Bars represent mean histology scores ± SD of six or seven mice per group pooled from two separate experiments except for the group receiving IL-10 KO CD4 + cells plus infected WT CD4 + cells ( n = 3), which was included in only one of the experiments. Statistical significance was tested for groups receiving IL-10 KO cells. *, P

Techniques Used: Infection, Mouse Assay

CD45RB low CD4 + cells from naive WT mice are unable to protect RAG KO recipients from colitis induced by pathogenic T cells plus H. hepaticus infection. (A) Infected RAG KO mice were given either no cells or CD4 + cells from infected IL-10 KO mice and CD45RB low CD4 + cells from naive or infected WT mice (4 × 10 5 of each population), and tissues were analyzed 4 wk later. Bars represent mean cecal (solid bars) and colonic (gray bars) histology scores ± SD of three mice per group. (B and C) Uninfected or H. hepaticus –infected (Hh-inf.) RAG KO mice were given either no cells or CD45RB hi CD4 + cells from naive WT mice and CD45RB low CD4 + cells from naive or infected WT mice (3 × 10 5 of each population). Body weights were measured weekly and tissues were analyzed after 4 and 7.5 wk for infected and uninfected recipients, respectively. (B) Results shown represent mean body weights (expressed as a percentage of the weight 2 d after cell transfer) of four mice per group from the uninfected RAG KO recipients. No difference in body weight was observed between the four groups of infected recipients over the 4-wk time period that these groups were being followed (not depicted). (C) Bars represent mean cecal (solid bars) and colonic (gray bars) histology scores ± SD of three or four mice per group. Statistical significance was tested for groups receiving pathogenic cells. *, P
Figure Legend Snippet: CD45RB low CD4 + cells from naive WT mice are unable to protect RAG KO recipients from colitis induced by pathogenic T cells plus H. hepaticus infection. (A) Infected RAG KO mice were given either no cells or CD4 + cells from infected IL-10 KO mice and CD45RB low CD4 + cells from naive or infected WT mice (4 × 10 5 of each population), and tissues were analyzed 4 wk later. Bars represent mean cecal (solid bars) and colonic (gray bars) histology scores ± SD of three mice per group. (B and C) Uninfected or H. hepaticus –infected (Hh-inf.) RAG KO mice were given either no cells or CD45RB hi CD4 + cells from naive WT mice and CD45RB low CD4 + cells from naive or infected WT mice (3 × 10 5 of each population). Body weights were measured weekly and tissues were analyzed after 4 and 7.5 wk for infected and uninfected recipients, respectively. (B) Results shown represent mean body weights (expressed as a percentage of the weight 2 d after cell transfer) of four mice per group from the uninfected RAG KO recipients. No difference in body weight was observed between the four groups of infected recipients over the 4-wk time period that these groups were being followed (not depicted). (C) Bars represent mean cecal (solid bars) and colonic (gray bars) histology scores ± SD of three or four mice per group. Statistical significance was tested for groups receiving pathogenic cells. *, P

Techniques Used: Mouse Assay, Infection

CD25 − CD45RB low and CD25 + CD45RB low CD4 + cells from infected WT mice block colitis via an IL-10–dependent but TGF-β–independent mechanism. Infected RAG KO mice were given either no cells or CD4 + cells from infected IL-10 KO mice and CD25 − CD45RB low or CD25 + CD45RB low CD4 + cells from infected WT mice (3 × 10 5 of each population) and treated with control mAb, anti–IL-10R (both at 1 mg/wk), or anti–TGF-β as indicated. (A) In the first experiment, mice were given 2 mg of anti–TGF-β per wk for 4 wk, (B) and in the second experiment, they were given 4 mg of anti–TGF-β per wk for 2 wk before analysis. •, the cecal histology score from an individual mouse; —, the average for each group. Similar results, although lower histology scores, were seen for the ascending colon where CD25 − cell recipients treated with control or anti–TGF-β mAb displayed indistinguishable scores in B. In A, anti–IL-10R–treated groups had P values of
Figure Legend Snippet: CD25 − CD45RB low and CD25 + CD45RB low CD4 + cells from infected WT mice block colitis via an IL-10–dependent but TGF-β–independent mechanism. Infected RAG KO mice were given either no cells or CD4 + cells from infected IL-10 KO mice and CD25 − CD45RB low or CD25 + CD45RB low CD4 + cells from infected WT mice (3 × 10 5 of each population) and treated with control mAb, anti–IL-10R (both at 1 mg/wk), or anti–TGF-β as indicated. (A) In the first experiment, mice were given 2 mg of anti–TGF-β per wk for 4 wk, (B) and in the second experiment, they were given 4 mg of anti–TGF-β per wk for 2 wk before analysis. •, the cecal histology score from an individual mouse; —, the average for each group. Similar results, although lower histology scores, were seen for the ascending colon where CD25 − cell recipients treated with control or anti–TGF-β mAb displayed indistinguishable scores in B. In A, anti–IL-10R–treated groups had P values of

Techniques Used: Infection, Mouse Assay, Blocking Assay

Intestinal pathology of H. hepaticus –infected RAG KO mice receiving CD4 + cells from IL-10 KO and WT mice. (A–F) Representative cecal sections of the infected RAG KO mice described in Fig. 2 A receiving (A) no cells, (B) infected IL-10 KO CD4 + cells, (C) infected IL-10 KO CD4 + cells plus infected WT CD4 + cells, (D) infected IL-10 KO CD4 + cells plus naive WT CD4 + cells, (E) infected WT CD4 + cells alone, and (F) naive WT CD4 + cells alone. H E staining. (G and H) Immunohistochemical staining for CD3 + T cells (arrows) in colonic sections of infected RAG KO recipients receiving (G) infected IL-10 KO CD4 + cells alone and (H) infected IL-10 KO CD4 + cells plus infected WT CD45RB low CD4 + cells. Bar, 200 μm.
Figure Legend Snippet: Intestinal pathology of H. hepaticus –infected RAG KO mice receiving CD4 + cells from IL-10 KO and WT mice. (A–F) Representative cecal sections of the infected RAG KO mice described in Fig. 2 A receiving (A) no cells, (B) infected IL-10 KO CD4 + cells, (C) infected IL-10 KO CD4 + cells plus infected WT CD4 + cells, (D) infected IL-10 KO CD4 + cells plus naive WT CD4 + cells, (E) infected WT CD4 + cells alone, and (F) naive WT CD4 + cells alone. H E staining. (G and H) Immunohistochemical staining for CD3 + T cells (arrows) in colonic sections of infected RAG KO recipients receiving (G) infected IL-10 KO CD4 + cells alone and (H) infected IL-10 KO CD4 + cells plus infected WT CD45RB low CD4 + cells. Bar, 200 μm.

Techniques Used: Infection, Mouse Assay, Staining, Immunohistochemistry

CD25 − CD45RB low as well as CD25 + CD45RB low CD4 + cells from infected WT mice protect RAG KO mice against colitis. Infected RAG KO mice were given either no cells or 3 × 10 5 CD4 + cells from infected IL-10 KO mice either alone or with 3 × 10 5 CD45RB low CD4 + cells, 3 × 10 5 , 10 5 , or 3.3 × 10 4 CD25 − CD45RB low , or CD25 + CD45RB low CD4 + cells from infected WT mice as indicated. (A) Pathology in the cecum and (B) ascending colon was analyzed 4 wk later. •, an individual mouse; —, the average for each group. Data shown are pooled from two separate experiments. *, P
Figure Legend Snippet: CD25 − CD45RB low as well as CD25 + CD45RB low CD4 + cells from infected WT mice protect RAG KO mice against colitis. Infected RAG KO mice were given either no cells or 3 × 10 5 CD4 + cells from infected IL-10 KO mice either alone or with 3 × 10 5 CD45RB low CD4 + cells, 3 × 10 5 , 10 5 , or 3.3 × 10 4 CD25 − CD45RB low , or CD25 + CD45RB low CD4 + cells from infected WT mice as indicated. (A) Pathology in the cecum and (B) ascending colon was analyzed 4 wk later. •, an individual mouse; —, the average for each group. Data shown are pooled from two separate experiments. *, P

Techniques Used: Infection, Mouse Assay

H. hepaticus –infected but not naive RAG KO mice develop intestinal inflammation after reconstitution with CD4 + T cells from IL-10 KO mice. (A) Uninfected or H. hepaticus –infected RAG KO mice were inoculated intravenously with 3 × 10 5 CD4 + cells from the MLNs of 11-wk infected IL-10 KO mice, and intestinal pathology was analyzed 8, 15, 22, and 29 d later (solid bars). Naive and infected RAG KO animals receiving no cells were analyzed in parallel (open bars). Bars represent mean cecal histology scores ± SD of three or four mice per group. *, P
Figure Legend Snippet: H. hepaticus –infected but not naive RAG KO mice develop intestinal inflammation after reconstitution with CD4 + T cells from IL-10 KO mice. (A) Uninfected or H. hepaticus –infected RAG KO mice were inoculated intravenously with 3 × 10 5 CD4 + cells from the MLNs of 11-wk infected IL-10 KO mice, and intestinal pathology was analyzed 8, 15, 22, and 29 d later (solid bars). Naive and infected RAG KO animals receiving no cells were analyzed in parallel (open bars). Bars represent mean cecal histology scores ± SD of three or four mice per group. *, P

Techniques Used: Infection, Mouse Assay

T cell–derived IL-10 mediates the disease-protective effect of WT CD45RB low CD4 + cells. (A) Infected RAG/IL-10 double deficient mice were given either no cells or infected IL-10 KO CD4 + cells and infected WT CD4 + CD45RB low cells as indicated (4 × 10 5 of each population) and then treated with control mAb or anti–IL-10R mAb for 4 wk. Bars represent mean cecal histology scores ± SD of three mice per group. (B) Infected RAG KO mice (solid bars) were inoculated with CD4 + cells from infected IL-10 KO, WT, and IL-4 KO mice as indicated (3 × 10 5 of each population) and then treated with control mAb or anti–IL-10R mAb for 4 wk. Naive (open bar) and infected RAG KO animals receiving no cells were included as controls. Bars represent mean cecal histology scores ± SD of three mice per group from one representative experiment out of two performed. Statistical significance was tested for groups receiving IL-10 KO cells. *, P
Figure Legend Snippet: T cell–derived IL-10 mediates the disease-protective effect of WT CD45RB low CD4 + cells. (A) Infected RAG/IL-10 double deficient mice were given either no cells or infected IL-10 KO CD4 + cells and infected WT CD4 + CD45RB low cells as indicated (4 × 10 5 of each population) and then treated with control mAb or anti–IL-10R mAb for 4 wk. Bars represent mean cecal histology scores ± SD of three mice per group. (B) Infected RAG KO mice (solid bars) were inoculated with CD4 + cells from infected IL-10 KO, WT, and IL-4 KO mice as indicated (3 × 10 5 of each population) and then treated with control mAb or anti–IL-10R mAb for 4 wk. Naive (open bar) and infected RAG KO animals receiving no cells were included as controls. Bars represent mean cecal histology scores ± SD of three mice per group from one representative experiment out of two performed. Statistical significance was tested for groups receiving IL-10 KO cells. *, P

Techniques Used: Derivative Assay, Infection, Mouse Assay

30) Product Images from "The survival of memory CD8 T cells that is mediated by IL-15 correlates with sustained protection against malaria 5The survival of memory CD8 T cells that is mediated by IL-15 correlates with sustained protection against malaria 5 6"

Article Title: The survival of memory CD8 T cells that is mediated by IL-15 correlates with sustained protection against malaria 5The survival of memory CD8 T cells that is mediated by IL-15 correlates with sustained protection against malaria 5 6

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

doi: 10.4049/jimmunol.1203396

IL-15KO CD8 T cells respond to antigen from Pb γ-spz Wt and IL-15KO mice (3–5 mice/group) were immunized with 3 weekly doses of Pb γ-spz followed one week later by infectious sporozoite challenge as described in materials and methods. (A) Livers from naïve, immune, or immune/challenged (CH) mice were isolated 1 week after last exposure to antigen and stained with anti-CD3, -CD8, -CD44, -CD62L mAbs to determine the number of total CD8 T cells, CD8 T E/EM cells and CD8 T CM cells. The results show the number of cells as the mean ± SD (B) IHMC were isolated from wt and IL-15KO mice one week following the 3 rd immunization with Pb γ-spz. IFN-γ was detected by cytokine secretion assay, as described in Materials and Methods. CD8 T cells were enriched by magnetic bead separation and CD44 hi CD45RB hi or CD44 hi CD45RB lo T cells were identified by flow cytometry. The results are representative of several experiments performed under the same conditions and show the number of IFN-γ secreting CD8 T CM cells and CD8 T E/EM cells per 10 6 IHMC determined for each individual mouse and are presented as the mean ± SD, *p
Figure Legend Snippet: IL-15KO CD8 T cells respond to antigen from Pb γ-spz Wt and IL-15KO mice (3–5 mice/group) were immunized with 3 weekly doses of Pb γ-spz followed one week later by infectious sporozoite challenge as described in materials and methods. (A) Livers from naïve, immune, or immune/challenged (CH) mice were isolated 1 week after last exposure to antigen and stained with anti-CD3, -CD8, -CD44, -CD62L mAbs to determine the number of total CD8 T cells, CD8 T E/EM cells and CD8 T CM cells. The results show the number of cells as the mean ± SD (B) IHMC were isolated from wt and IL-15KO mice one week following the 3 rd immunization with Pb γ-spz. IFN-γ was detected by cytokine secretion assay, as described in Materials and Methods. CD8 T cells were enriched by magnetic bead separation and CD44 hi CD45RB hi or CD44 hi CD45RB lo T cells were identified by flow cytometry. The results are representative of several experiments performed under the same conditions and show the number of IFN-γ secreting CD8 T CM cells and CD8 T E/EM cells per 10 6 IHMC determined for each individual mouse and are presented as the mean ± SD, *p

Techniques Used: Mouse Assay, Isolation, Staining, Flow Cytometry, Cytometry

CD8 T CM cells, having elevated expression of IL-15Rβ (CD122), are the primary cells responding to IL-15 IHMC were isolated from C57BL/6 mice (3 per group) 1 month after a tertiary immunization with Pb γ-spz. (A) CD62L + and CD62L − T cells were separated by magnetic bead isolation procedure; a second round of isolation using CD8 magnetic beads resulted in the following subpopulations: CD8 + CD62L + and CD8 + CD62L − T cells as well as CD8 − CD62L + and CD8 − CD62L − T cells. Each T cell subset was cultured at 4 × 10 5 cells/0.2 ml culture medium for 96 hours in the presence of the indicted concentrations of IL-15; during the last 16hrs of culture, 1μCi of 3 H-TdR was added to each well. Results are presented as the mean CPM ± SD of 3 H-TdR uptake in triplicate wells and are representative of 3 separate experiments. (B) Liver CD8 T cells, isolated by negative magnetic beads selection, were stained with CFSE and incubated for 7 days in the presence of 100 ng/ml IL-15, or 100ng/ml IL-2. Harvested cells were stained with mAbs against CD8, CD44 and CD45RB. Lymphocytes were gated on a forward-side scatter plot, and gates were applied to identify CD8 + T cells that expressed either CD44 hi CD45RB hi (T CM cells) or CD44 hi CD45RB lo (T E/EM cells) phenotypes. Histogram plots represent CFSE dilution with gray shaded area representing CD8 T CM cells and solid contour line representing CD8 T E/EM cells. These results represent one of two separate experiments. (C) Liver CD8 T cells were cultured in the presence of either 100 ng/ml IL-15, or medium alone. Cells were harvested on day 1, 3, and 7 and stained with mAbs against CD44 and CD45RB as described above, fixed, permeabilized and stained with mAbs against Bcl-2. Mean fluorescent intensity (determined for each individual mouse and is presented as the mean ± SD) of Bcl-2 expression of T CM or T E/EM . Bcl-2 determinations on CD8 T CM and CD8 T E/EM cells prior to culture showed MFI=189± 45 for CD8 T CM and MFI= 76± 35 for CD8 T E/EM . Results are representative of 3 separate experiments. *p
Figure Legend Snippet: CD8 T CM cells, having elevated expression of IL-15Rβ (CD122), are the primary cells responding to IL-15 IHMC were isolated from C57BL/6 mice (3 per group) 1 month after a tertiary immunization with Pb γ-spz. (A) CD62L + and CD62L − T cells were separated by magnetic bead isolation procedure; a second round of isolation using CD8 magnetic beads resulted in the following subpopulations: CD8 + CD62L + and CD8 + CD62L − T cells as well as CD8 − CD62L + and CD8 − CD62L − T cells. Each T cell subset was cultured at 4 × 10 5 cells/0.2 ml culture medium for 96 hours in the presence of the indicted concentrations of IL-15; during the last 16hrs of culture, 1μCi of 3 H-TdR was added to each well. Results are presented as the mean CPM ± SD of 3 H-TdR uptake in triplicate wells and are representative of 3 separate experiments. (B) Liver CD8 T cells, isolated by negative magnetic beads selection, were stained with CFSE and incubated for 7 days in the presence of 100 ng/ml IL-15, or 100ng/ml IL-2. Harvested cells were stained with mAbs against CD8, CD44 and CD45RB. Lymphocytes were gated on a forward-side scatter plot, and gates were applied to identify CD8 + T cells that expressed either CD44 hi CD45RB hi (T CM cells) or CD44 hi CD45RB lo (T E/EM cells) phenotypes. Histogram plots represent CFSE dilution with gray shaded area representing CD8 T CM cells and solid contour line representing CD8 T E/EM cells. These results represent one of two separate experiments. (C) Liver CD8 T cells were cultured in the presence of either 100 ng/ml IL-15, or medium alone. Cells were harvested on day 1, 3, and 7 and stained with mAbs against CD44 and CD45RB as described above, fixed, permeabilized and stained with mAbs against Bcl-2. Mean fluorescent intensity (determined for each individual mouse and is presented as the mean ± SD) of Bcl-2 expression of T CM or T E/EM . Bcl-2 determinations on CD8 T CM and CD8 T E/EM cells prior to culture showed MFI=189± 45 for CD8 T CM and MFI= 76± 35 for CD8 T E/EM . Results are representative of 3 separate experiments. *p

Techniques Used: Expressing, Isolation, Mouse Assay, Magnetic Beads, Cell Culture, Selection, Staining, Incubation

31) Product Images from "Distinct Roles for CXCR6+ and CXCR6− CD4+ T Cells in the Pathogenesis of Chronic Colitis"

Article Title: Distinct Roles for CXCR6+ and CXCR6− CD4+ T Cells in the Pathogenesis of Chronic Colitis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0065488

CXCR6 expression is not required for development of transfer colitis. ( A ) Body weight of Rag1 −/− recipients of i.v. injected purified CD45RB high CD4 + T cells form Cxcr6 +/Egfp or Cxcr6 Egfp/Egfp (CXCR6-deficient) mice on day 0, presented as percent of original weight. ( B ) Colon weight of the mice in ( A ) on week 7. Data are representative of two independent experiments (mean and s.d.). ( C ) Histology of colon tissues from the mice in B . ( D ) CXCR6 expression by LP CD4 + T cells was analyzed by flow cytometry using CXCL16-hFc at 7-week post transfer. ( E ) Expression levels of indicated cytokines in distal colon were analyzed by Q-PCR at 7 weeks after the transfer. Data were normalized to expression of Gapdh . ( n = 4 or 5; mean and s.d.).
Figure Legend Snippet: CXCR6 expression is not required for development of transfer colitis. ( A ) Body weight of Rag1 −/− recipients of i.v. injected purified CD45RB high CD4 + T cells form Cxcr6 +/Egfp or Cxcr6 Egfp/Egfp (CXCR6-deficient) mice on day 0, presented as percent of original weight. ( B ) Colon weight of the mice in ( A ) on week 7. Data are representative of two independent experiments (mean and s.d.). ( C ) Histology of colon tissues from the mice in B . ( D ) CXCR6 expression by LP CD4 + T cells was analyzed by flow cytometry using CXCL16-hFc at 7-week post transfer. ( E ) Expression levels of indicated cytokines in distal colon were analyzed by Q-PCR at 7 weeks after the transfer. Data were normalized to expression of Gapdh . ( n = 4 or 5; mean and s.d.).

Techniques Used: Expressing, Injection, Purification, Mouse Assay, Flow Cytometry, Cytometry, Polymerase Chain Reaction

CXCR6 expression is related to the production of Th1 and Th17 cytokines. Intracellular staining for cytokine and transcription factors in CD4 + T cells was performed 8 weeks after naïve CD4 + T-cell transfer. ( A, B ) The frequency of IL-2 + cells and IFN-γ + cells was analyzed in the CXCR6 − (upper) and CXCR6 + (lower) subsets ( A ) and the absolute number of T cells were graphed on the basis of the flow cytometric analysis ( B ). ( C, D ) The frequency of IL-17A + cells and TNF-α + cells was analyzed in CXCR6 − (upper) and CXCR6 + (lower) subset ( C ) and the numbers were graphed on the basis of flow cytometric analysis ( D ). ( E ) The frequency of IFN-γ + cells and IL-17A + cells was analyzed. ( F ) The frequency of T-bet + cells and RORγt + cells was analyzed. All data are representative from four independent experiments (mean and s.d.). *, P
Figure Legend Snippet: CXCR6 expression is related to the production of Th1 and Th17 cytokines. Intracellular staining for cytokine and transcription factors in CD4 + T cells was performed 8 weeks after naïve CD4 + T-cell transfer. ( A, B ) The frequency of IL-2 + cells and IFN-γ + cells was analyzed in the CXCR6 − (upper) and CXCR6 + (lower) subsets ( A ) and the absolute number of T cells were graphed on the basis of the flow cytometric analysis ( B ). ( C, D ) The frequency of IL-17A + cells and TNF-α + cells was analyzed in CXCR6 − (upper) and CXCR6 + (lower) subset ( C ) and the numbers were graphed on the basis of flow cytometric analysis ( D ). ( E ) The frequency of IFN-γ + cells and IL-17A + cells was analyzed. ( F ) The frequency of T-bet + cells and RORγt + cells was analyzed. All data are representative from four independent experiments (mean and s.d.). *, P

Techniques Used: Expressing, Staining, Flow Cytometry

LP CXCR6 − but not CXCR6 + cells can transfer wasting and colitis upon retransfer into Rag1 −/− recipients. ( A ) Schematic transfer protocol. An equal number of CD25 − CXCR6 − cells or CD25 − CXCR6 + CD4 + T cell isolated from colon LP of colitic mice was retransferred into Rag1 −/− mice. ( B ) Time course of changes in body weight after retransfer of the two subsets or transfer of CD45RB high naïve T cells. CXCR6 – transferred Rag1 −/− mice manifested progressive body weight loss to a similar extent to CD45RB high -transferred Rag1 −/− mice, whereas the cohort that received CXCR6 + cells did not show wasting. Data are expressed as the mean and s.e.m of two independent experiments. *, P
Figure Legend Snippet: LP CXCR6 − but not CXCR6 + cells can transfer wasting and colitis upon retransfer into Rag1 −/− recipients. ( A ) Schematic transfer protocol. An equal number of CD25 − CXCR6 − cells or CD25 − CXCR6 + CD4 + T cell isolated from colon LP of colitic mice was retransferred into Rag1 −/− mice. ( B ) Time course of changes in body weight after retransfer of the two subsets or transfer of CD45RB high naïve T cells. CXCR6 – transferred Rag1 −/− mice manifested progressive body weight loss to a similar extent to CD45RB high -transferred Rag1 −/− mice, whereas the cohort that received CXCR6 + cells did not show wasting. Data are expressed as the mean and s.e.m of two independent experiments. *, P

Techniques Used: Isolation, Mouse Assay

CXCR6 is expressed both by the effector and effector memory CD4 + T cells in the inflamed colon. LP CXCR6 − and CXCR6 + cells were analyzed for expression of activation and memory markers at week 8 post-transfer of naïve CD4 + T cells. ( A – C ) CD4 + T cells were gated as CD127 − CD62L − CD27 − CD43 + CD44 + to measure the proportion of effector T cells (a). ( D – F ) The effector memory population (CD44 + CD127 + ) was subdivided using CD62L and CD27 to measure early effector memory cells (CD62L − CD27 + CD43 + , b) and late effector memory cells (CD62L − CD27 − CD43 + , c). Data are representative of three independent experiments. ( G ) The relative percentages of effector, early effector memory and late effector memory in each subset are shown in a pie chart.
Figure Legend Snippet: CXCR6 is expressed both by the effector and effector memory CD4 + T cells in the inflamed colon. LP CXCR6 − and CXCR6 + cells were analyzed for expression of activation and memory markers at week 8 post-transfer of naïve CD4 + T cells. ( A – C ) CD4 + T cells were gated as CD127 − CD62L − CD27 − CD43 + CD44 + to measure the proportion of effector T cells (a). ( D – F ) The effector memory population (CD44 + CD127 + ) was subdivided using CD62L and CD27 to measure early effector memory cells (CD62L − CD27 + CD43 + , b) and late effector memory cells (CD62L − CD27 − CD43 + , c). Data are representative of three independent experiments. ( G ) The relative percentages of effector, early effector memory and late effector memory in each subset are shown in a pie chart.

Techniques Used: Expressing, Activation Assay

32) Product Images from "Amiselimod (MT-1303), a novel sphingosine 1-phosphate receptor-1 functional antagonist, inhibits progress of chronic colitis induced by transfer of CD4+CD45RBhigh T cells"

Article Title: Amiselimod (MT-1303), a novel sphingosine 1-phosphate receptor-1 functional antagonist, inhibits progress of chronic colitis induced by transfer of CD4+CD45RBhigh T cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0226154

Effect of MT-1303 on infiltration of Th1 and Th17 cells into the lamina propria of the colon. MT-1303 or vehicle was orally administered to SCID mice every day from week 1 after CD4 + CD45RB high T cell transfer for 3–4 weeks. (A, B) The number of lymphocytes (A) and CD4 + T cells (B) in the lamina propria of the colon was measured using flow cytometry the day after the final administration. (C–E) LPLs were stimulated with PMA and ionomycin in the presence of brefeldin A for 6 h before intracellular cytokine staining was performed using anti-IFN-γ and anti-IL-17 mAbs (C). Th1 (D) and Th17 (E) cell counts in the lamina propria of the colon were determined. (F, G) After stimulation with PMA and ionomycin, the amount of IFN-γ (F) and IL-17 (G) in the culture supernatant was determined using a BD TM Cytometric Beads Array. Each bar represents the mean ± S.E.M. (n = 11: vehicle-treated group, n = 12: MT-1303-treated group). Statistical differences were determined using Student’s t -test by comparing with the normal group (* p
Figure Legend Snippet: Effect of MT-1303 on infiltration of Th1 and Th17 cells into the lamina propria of the colon. MT-1303 or vehicle was orally administered to SCID mice every day from week 1 after CD4 + CD45RB high T cell transfer for 3–4 weeks. (A, B) The number of lymphocytes (A) and CD4 + T cells (B) in the lamina propria of the colon was measured using flow cytometry the day after the final administration. (C–E) LPLs were stimulated with PMA and ionomycin in the presence of brefeldin A for 6 h before intracellular cytokine staining was performed using anti-IFN-γ and anti-IL-17 mAbs (C). Th1 (D) and Th17 (E) cell counts in the lamina propria of the colon were determined. (F, G) After stimulation with PMA and ionomycin, the amount of IFN-γ (F) and IL-17 (G) in the culture supernatant was determined using a BD TM Cytometric Beads Array. Each bar represents the mean ± S.E.M. (n = 11: vehicle-treated group, n = 12: MT-1303-treated group). Statistical differences were determined using Student’s t -test by comparing with the normal group (* p

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Staining

33) Product Images from "Expanded B cell population blocks regulatory T cells and exacerbates ileitis in a murine model of Crohn disease"

Article Title: Expanded B cell population blocks regulatory T cells and exacerbates ileitis in a murine model of Crohn disease

Journal: Journal of Clinical Investigation

doi: 10.1172/JCI200420855

Soluble IgA production increased in SAMP1/YitFc versus AKR mice. ( A ) Representative dot plots of κ (1:100 dilution, FL2) and λ (1:1, FL1) light chain antibody isotype levels within MLN supernatants from AKR mice ( n = 2) and SAMP1/YitFc mice ( n = 2), measured with a cytometric bead array containing isotype-specific beads with preset FL3 intensities. MLNs were crushed, resuspended in 5 ml, and centrifuged to remove the cell pellet from the tested supernatants. An increase of at least twofold in IgG1 κ, IgA κ, IgM κ, IgA λ, and IgM λ was seen in SAMP1/YitFc versus AKR MLNs, as measured by mean fluorescence intensity of the isotype-specific beads. ( B ) ELISA detecting IgA antibody concentrations (mean ± SEM) in serum samples collected via cardiac puncture from SAMP1/YitFc mice ( n = 12) and wild-type AKR mice ( n = 13). ( C ) Concentrations of IgA ( n = 4), IgM ( n = 2), and IgG2a ( n = 2), measured in triplicate by ELISA, from 3-, 7-, and 11-day anti-CD3–stimulated cocultures of SAMP1/YitFc CD4 + T cells and B cells versus AKR CD4 + T cells and B cells (105 T cells/well and 105 B cells/well). *Significantly greater than AKR concentrations ( P
Figure Legend Snippet: Soluble IgA production increased in SAMP1/YitFc versus AKR mice. ( A ) Representative dot plots of κ (1:100 dilution, FL2) and λ (1:1, FL1) light chain antibody isotype levels within MLN supernatants from AKR mice ( n = 2) and SAMP1/YitFc mice ( n = 2), measured with a cytometric bead array containing isotype-specific beads with preset FL3 intensities. MLNs were crushed, resuspended in 5 ml, and centrifuged to remove the cell pellet from the tested supernatants. An increase of at least twofold in IgG1 κ, IgA κ, IgM κ, IgA λ, and IgM λ was seen in SAMP1/YitFc versus AKR MLNs, as measured by mean fluorescence intensity of the isotype-specific beads. ( B ) ELISA detecting IgA antibody concentrations (mean ± SEM) in serum samples collected via cardiac puncture from SAMP1/YitFc mice ( n = 12) and wild-type AKR mice ( n = 13). ( C ) Concentrations of IgA ( n = 4), IgM ( n = 2), and IgG2a ( n = 2), measured in triplicate by ELISA, from 3-, 7-, and 11-day anti-CD3–stimulated cocultures of SAMP1/YitFc CD4 + T cells and B cells versus AKR CD4 + T cells and B cells (105 T cells/well and 105 B cells/well). *Significantly greater than AKR concentrations ( P

Techniques Used: Mouse Assay, Fluorescence, Enzyme-linked Immunosorbent Assay

34) Product Images from "Hsp65-Producing Lactococcus lactis Prevents Inflammatory Intestinal Disease in Mice by IL-10- and TLR2-Dependent Pathways"

Article Title: Hsp65-Producing Lactococcus lactis Prevents Inflammatory Intestinal Disease in Mice by IL-10- and TLR2-Dependent Pathways

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00030

Hsp65-producing L. lactis (Hsp65-LL) induces increased frequencies of regulatory T cells (Tregs) in mice . C57BL/6 mice were pretreated or not (naïve) with medium (CT), empty vector-harboring L. lactis (CT-LL), or Hsp65-LL for 4 days and colitis was induced 10 days later by 1.5% DSS in drinking water. Seventy-two hours after DSS administration, mice were sacrificed and mesenteric lymph nodes (mLNs) removed. Cells were stained with fluorescein isothiocyanate-conjugated (FITC)-anti-CD4, PE-anti-Foxp3, Bio-CD25 and CY5.5-STV (A) or with FITC-anti-CD4, PE-anti-CD25, Bio-LAP, and Cy5.5-STV (B) . Alternatively, C57BL/6 mice were orally pretreated or not (naïve) with Hsp65-LL during 4 days. Either 4 or 10 days after the last day of oral treatment frequencies of Treg populations were analyzed by flow cytometry. (C) Spleen and (D) mLN cells were stained with Cy5.5 anti-CD4 and PE anti-Foxp3. (E) Spleen cells were stained with Cy5.5 anti-CD4; biotin anti-LAP, and APC streptavidin. CD4 + Foxp3 + and CD4 + LAP + cells were gated on lymphocyte population ( N = 4). Results are representative of three independent experiments. Bar graphs are shown as mean ± SEM. Analysis of variance, Tukey’s post hoc test, p
Figure Legend Snippet: Hsp65-producing L. lactis (Hsp65-LL) induces increased frequencies of regulatory T cells (Tregs) in mice . C57BL/6 mice were pretreated or not (naïve) with medium (CT), empty vector-harboring L. lactis (CT-LL), or Hsp65-LL for 4 days and colitis was induced 10 days later by 1.5% DSS in drinking water. Seventy-two hours after DSS administration, mice were sacrificed and mesenteric lymph nodes (mLNs) removed. Cells were stained with fluorescein isothiocyanate-conjugated (FITC)-anti-CD4, PE-anti-Foxp3, Bio-CD25 and CY5.5-STV (A) or with FITC-anti-CD4, PE-anti-CD25, Bio-LAP, and Cy5.5-STV (B) . Alternatively, C57BL/6 mice were orally pretreated or not (naïve) with Hsp65-LL during 4 days. Either 4 or 10 days after the last day of oral treatment frequencies of Treg populations were analyzed by flow cytometry. (C) Spleen and (D) mLN cells were stained with Cy5.5 anti-CD4 and PE anti-Foxp3. (E) Spleen cells were stained with Cy5.5 anti-CD4; biotin anti-LAP, and APC streptavidin. CD4 + Foxp3 + and CD4 + LAP + cells were gated on lymphocyte population ( N = 4). Results are representative of three independent experiments. Bar graphs are shown as mean ± SEM. Analysis of variance, Tukey’s post hoc test, p

Techniques Used: Mouse Assay, Plasmid Preparation, Staining, Flow Cytometry, Cytometry

Anti-colitogenic effect of Hsp65-producing L. lactis (Hsp65-LL) is dependent on IL-10 . Groups of 6-week-old IL-10-deficient (IL-10 −/− ) mice or control (129Sv/Ev) mice were pretreated or not (naïve) with medium (CT) or Hsp65-LL for four consecutive days. Ten days later, mice were given 1.5% DSS in drinking water for 7 days. (A) Macroscopic score including body weight loss, stool consistency, and bleeding were scored at day 7. (B) At day 7, colons were collected and dissected for histological analysis by hematoxylin and eosin staining. Histological scores (sum of loss of mucosal architecture, cellular infiltration, muscle thickening, crypt abscess formation, and goblet cell depletion) were ranked blindly ( N = 4). (C,D) Groups of 6-week-old IL-10-deficient mice were pretreated or not (naïve) with medium (CT), empty vector-harboring L. lactis (CT-LL), or Hsp65-LL for four consecutive days. When IL-10 −/− mice completed 12 weeks of age, macroscopic and histological scores were ranked. (E) Cytokines such as IFN-γ, IL-17, and IL-10 were measured in colon extracts at the end of the experiment. (F) Lamina propria (LP) T cells from entire colon, cells isolated from mesenteric lymph nodes (mLNs) or from cecal lymph nodes (cLN) were obtained from IL-10 −/− mice. Frequency of CD4 + CD25 + LAP + T cells was assessed by flow cytometry and gated in the lymphocyte population ( N = 8). Results are representatives of two independent experiments. Bar graphs are shown as mean ± SEM. Analysis of variance, Tukey’s post hoc test, p
Figure Legend Snippet: Anti-colitogenic effect of Hsp65-producing L. lactis (Hsp65-LL) is dependent on IL-10 . Groups of 6-week-old IL-10-deficient (IL-10 −/− ) mice or control (129Sv/Ev) mice were pretreated or not (naïve) with medium (CT) or Hsp65-LL for four consecutive days. Ten days later, mice were given 1.5% DSS in drinking water for 7 days. (A) Macroscopic score including body weight loss, stool consistency, and bleeding were scored at day 7. (B) At day 7, colons were collected and dissected for histological analysis by hematoxylin and eosin staining. Histological scores (sum of loss of mucosal architecture, cellular infiltration, muscle thickening, crypt abscess formation, and goblet cell depletion) were ranked blindly ( N = 4). (C,D) Groups of 6-week-old IL-10-deficient mice were pretreated or not (naïve) with medium (CT), empty vector-harboring L. lactis (CT-LL), or Hsp65-LL for four consecutive days. When IL-10 −/− mice completed 12 weeks of age, macroscopic and histological scores were ranked. (E) Cytokines such as IFN-γ, IL-17, and IL-10 were measured in colon extracts at the end of the experiment. (F) Lamina propria (LP) T cells from entire colon, cells isolated from mesenteric lymph nodes (mLNs) or from cecal lymph nodes (cLN) were obtained from IL-10 −/− mice. Frequency of CD4 + CD25 + LAP + T cells was assessed by flow cytometry and gated in the lymphocyte population ( N = 8). Results are representatives of two independent experiments. Bar graphs are shown as mean ± SEM. Analysis of variance, Tukey’s post hoc test, p

Techniques Used: Mouse Assay, Staining, Plasmid Preparation, Isolation, Flow Cytometry, Cytometry

35) Product Images from "CD4+ T cell vaccination overcomes defective cross-presentation of fungal antigens in a mouse model of chronic granulomatous disease"

Article Title: CD4+ T cell vaccination overcomes defective cross-presentation of fungal antigens in a mouse model of chronic granulomatous disease

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI60862

CD11b + p47 phox–/– DCs efficiently present fungal antigens. ( A and B ) Flow cytometry of phagocytosis of live GFP-expressing conidia by lung DCs purified from uninfected mice. Values are percentages of positive cells on T and B cell–depleted lung cells. Phagocytosis was quantified via phase contrast and fluorescence microscopy at ×1,000 magnification (shown are representative microscopy images of 2 independent experiments). ( C ) Mice were infected i.n. with GFP-expressing A. fumigatus conidia, and the numbers of GFP + CD11c + cells were assessed by flow cytometry at 3 days after infection. Values are percentages of positive cells on gated CD11c + cells. DCs purified from lung of naive mice were unpulsed (None) or pulsed with live Aspergillus conidia or Pep1p for ( D ) 24 hours prior to being assessed for cytokine gene expression by RT-PCR and ( E ) 2 hours before the assessment of pERK44/42 phosphorylation by immunoblot analysis. Shown is a representative Western blot of 2 independent experiments and corresponding pixel density ratio normalized against β-tubulin (β-tub). ( F and G ) Adoptive transfer of C57BL/6 or p47 phox–/– lung DCs pulsed with conidia or Pep1p into C57BL/6 mice. DCs were adoptively transferred by i.p. injection twice, a week apart, before the i.n. infection. Fungal growth in the lungs ( F , log 10 CFU ± SEM, representative of at least 3 independent experiments) and histology ( G , PAS staining) were determined 3 days after infection. * P
Figure Legend Snippet: CD11b + p47 phox–/– DCs efficiently present fungal antigens. ( A and B ) Flow cytometry of phagocytosis of live GFP-expressing conidia by lung DCs purified from uninfected mice. Values are percentages of positive cells on T and B cell–depleted lung cells. Phagocytosis was quantified via phase contrast and fluorescence microscopy at ×1,000 magnification (shown are representative microscopy images of 2 independent experiments). ( C ) Mice were infected i.n. with GFP-expressing A. fumigatus conidia, and the numbers of GFP + CD11c + cells were assessed by flow cytometry at 3 days after infection. Values are percentages of positive cells on gated CD11c + cells. DCs purified from lung of naive mice were unpulsed (None) or pulsed with live Aspergillus conidia or Pep1p for ( D ) 24 hours prior to being assessed for cytokine gene expression by RT-PCR and ( E ) 2 hours before the assessment of pERK44/42 phosphorylation by immunoblot analysis. Shown is a representative Western blot of 2 independent experiments and corresponding pixel density ratio normalized against β-tubulin (β-tub). ( F and G ) Adoptive transfer of C57BL/6 or p47 phox–/– lung DCs pulsed with conidia or Pep1p into C57BL/6 mice. DCs were adoptively transferred by i.p. injection twice, a week apart, before the i.n. infection. Fungal growth in the lungs ( F , log 10 CFU ± SEM, representative of at least 3 independent experiments) and histology ( G , PAS staining) were determined 3 days after infection. * P

Techniques Used: Flow Cytometry, Cytometry, Expressing, Purification, Mouse Assay, Fluorescence, Microscopy, Infection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Adoptive Transfer Assay, Injection, Staining

36) Product Images from "IL-10-producing CD4+ T cells negatively regulate fucosylation of epithelial cells in the gut"

Article Title: IL-10-producing CD4+ T cells negatively regulate fucosylation of epithelial cells in the gut

Journal: Scientific Reports

doi: 10.1038/srep15918

Effector T cells are involved in the downregulation of epithelial fucosylation in the ileum. CD4 + CD45RB hi or CD4 + CD45RB lo T cells were purified from the spleens of C57BL/6 mice and adoptively transferred into Tcrb −/− mice. After 4 weeks, ileal ECs were analyzed for fucosylation by flow cytometry ( A ) and for Fut2 expression by quantitative PCR ( B ). Data in ( A ) are representative of 6 independent experiments. Data in ( B ) are shown as mean ± s.d. ( n = 6 from 2 independent experiments).
Figure Legend Snippet: Effector T cells are involved in the downregulation of epithelial fucosylation in the ileum. CD4 + CD45RB hi or CD4 + CD45RB lo T cells were purified from the spleens of C57BL/6 mice and adoptively transferred into Tcrb −/− mice. After 4 weeks, ileal ECs were analyzed for fucosylation by flow cytometry ( A ) and for Fut2 expression by quantitative PCR ( B ). Data in ( A ) are representative of 6 independent experiments. Data in ( B ) are shown as mean ± s.d. ( n = 6 from 2 independent experiments).

Techniques Used: Purification, Mouse Assay, Flow Cytometry, Cytometry, Expressing, Real-time Polymerase Chain Reaction

37) Product Images from "CARD15/NOD2 Is Required for Peyer's Patches Homeostasis in Mice"

Article Title: CARD15/NOD2 Is Required for Peyer's Patches Homeostasis in Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0000523

Nod2 and Peyer's patch cellular composition. (A and B) Relative proportions of T-, B- and dendritic cells from PPs and spleens of KO (▪) and WT (□) mice at week 12. T-, B- and dendritic cells were investigated by flow cytometry using antibodies to CD3, B220 and CD11c. (C) Relative proportion of polymorphonuclear neutrophils was analyzed using Ly-6G antibody. (D) M cells number inside the follicle associated with epithelium was investigated by immuno-histochemistry. Arrows indicated the presence of M-cell inside the follicle associated with epithelium. Data represent the means±SEM of 8 mice per group. **P
Figure Legend Snippet: Nod2 and Peyer's patch cellular composition. (A and B) Relative proportions of T-, B- and dendritic cells from PPs and spleens of KO (▪) and WT (□) mice at week 12. T-, B- and dendritic cells were investigated by flow cytometry using antibodies to CD3, B220 and CD11c. (C) Relative proportion of polymorphonuclear neutrophils was analyzed using Ly-6G antibody. (D) M cells number inside the follicle associated with epithelium was investigated by immuno-histochemistry. Arrows indicated the presence of M-cell inside the follicle associated with epithelium. Data represent the means±SEM of 8 mice per group. **P

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Immunohistochemistry

38) Product Images from "Amiselimod (MT-1303), a novel sphingosine 1-phosphate receptor-1 functional antagonist, inhibits progress of chronic colitis induced by transfer of CD4+CD45RBhigh T cells"

Article Title: Amiselimod (MT-1303), a novel sphingosine 1-phosphate receptor-1 functional antagonist, inhibits progress of chronic colitis induced by transfer of CD4+CD45RBhigh T cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0226154

Effect of MT-1303 on infiltration of Th1 and Th17 cells into the lamina propria of the colon. MT-1303 or vehicle was orally administered to SCID mice every day from week 1 after CD4 + CD45RB high T cell transfer for 3–4 weeks. (A, B) The number of lymphocytes (A) and CD4 + T cells (B) in the lamina propria of the colon was measured using flow cytometry the day after the final administration. (C–E) LPLs were stimulated with PMA and ionomycin in the presence of brefeldin A for 6 h before intracellular cytokine staining was performed using anti-IFN-γ and anti-IL-17 mAbs (C). Th1 (D) and Th17 (E) cell counts in the lamina propria of the colon were determined. (F, G) After stimulation with PMA and ionomycin, the amount of IFN-γ (F) and IL-17 (G) in the culture supernatant was determined using a BD TM Cytometric Beads Array. Each bar represents the mean ± S.E.M. (n = 11: vehicle-treated group, n = 12: MT-1303-treated group). Statistical differences were determined using Student’s t -test by comparing with the normal group (* p
Figure Legend Snippet: Effect of MT-1303 on infiltration of Th1 and Th17 cells into the lamina propria of the colon. MT-1303 or vehicle was orally administered to SCID mice every day from week 1 after CD4 + CD45RB high T cell transfer for 3–4 weeks. (A, B) The number of lymphocytes (A) and CD4 + T cells (B) in the lamina propria of the colon was measured using flow cytometry the day after the final administration. (C–E) LPLs were stimulated with PMA and ionomycin in the presence of brefeldin A for 6 h before intracellular cytokine staining was performed using anti-IFN-γ and anti-IL-17 mAbs (C). Th1 (D) and Th17 (E) cell counts in the lamina propria of the colon were determined. (F, G) After stimulation with PMA and ionomycin, the amount of IFN-γ (F) and IL-17 (G) in the culture supernatant was determined using a BD TM Cytometric Beads Array. Each bar represents the mean ± S.E.M. (n = 11: vehicle-treated group, n = 12: MT-1303-treated group). Statistical differences were determined using Student’s t -test by comparing with the normal group (* p

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Staining

Prophylactic effects of MT-1303 on colitis in SCID mice induced by adoptive transfer of CD4 + CD45RB high T cells. SCID mice were injected with CD4 + CD45RB high T cells to induce colitis. MT-1303 or vehicle was orally administered to SCID mice every day from the day of CD4 + CD45RB high T cell transfer for 28 days. (A) Change in body weight over time. Body weight on each day is expressed as a percentage of the original weight. Each symbol represents the mean ± S.E.M. of body weight (%) in 14–15 mice (n = 14: normal group). Statistical differences were determined using Student’s t -test by comparing to the normal group (# p
Figure Legend Snippet: Prophylactic effects of MT-1303 on colitis in SCID mice induced by adoptive transfer of CD4 + CD45RB high T cells. SCID mice were injected with CD4 + CD45RB high T cells to induce colitis. MT-1303 or vehicle was orally administered to SCID mice every day from the day of CD4 + CD45RB high T cell transfer for 28 days. (A) Change in body weight over time. Body weight on each day is expressed as a percentage of the original weight. Each symbol represents the mean ± S.E.M. of body weight (%) in 14–15 mice (n = 14: normal group). Statistical differences were determined using Student’s t -test by comparing to the normal group (# p

Techniques Used: Mouse Assay, Adoptive Transfer Assay, Injection

Effects of MT-1303 and an anti-mTNF-α mAb on colitis in SCID mice induced by adoptive transfer of CD4 + CD45RB high T cells. SCID mice were injected with CD4 + CD45RB high T cells to induce colitis. MT-1303 was orally administered to SCID mice every day from day 7 after CD4 + CD45RB high T cell transfer for 21 days, and anti-mTNF-α mAb was intraperitoneally administered to SCID mice on day 7 and day 21 after cell transfer. (A, B) Change in body weight over time. Body weight on each day is expressed as a percentage of the original weight. Each symbol represents the mean ± S.E.M. of body weight (%) in 18 mice. Statistical differences were determined using Student’s t -test by comparing with the normal group (# p
Figure Legend Snippet: Effects of MT-1303 and an anti-mTNF-α mAb on colitis in SCID mice induced by adoptive transfer of CD4 + CD45RB high T cells. SCID mice were injected with CD4 + CD45RB high T cells to induce colitis. MT-1303 was orally administered to SCID mice every day from day 7 after CD4 + CD45RB high T cell transfer for 21 days, and anti-mTNF-α mAb was intraperitoneally administered to SCID mice on day 7 and day 21 after cell transfer. (A, B) Change in body weight over time. Body weight on each day is expressed as a percentage of the original weight. Each symbol represents the mean ± S.E.M. of body weight (%) in 18 mice. Statistical differences were determined using Student’s t -test by comparing with the normal group (# p

Techniques Used: Mouse Assay, Adoptive Transfer Assay, Injection

39) Product Images from "Enhanced susceptibility to chemically induced colitis caused by excessive endosomal TLR signaling in LRBA-deficient mice"

Article Title: Enhanced susceptibility to chemically induced colitis caused by excessive endosomal TLR signaling in LRBA-deficient mice

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1901407116

DCs promote DSS-induced colitis in Lrba −/− mice. ( A – F ) BM transplantation was performed using mice of the indicated genotypes (donor > recipient). ( A and C ) Body weight (% relative to weight on day 0) on day 10 ( A ) or on the indicated day ( C ) after initiation of DSS treatment. Lrba +/+ > Lrba +/+ ( n = 5), Lrba −/− > Lrba +/+ ( n = 4), Lrba +/+ > Lrba −/− ( n = 3), Lrba −/− > Lrba −/− ( n = 5) for A. Lrba +/+ Rag2 −/− > Rag2 −/− ( n = 4), Lrba −/− Rag2 −/− > Rag2 −/− ( n = 4) for C. ( B and D ) DAI and ( E ) colon length on day 10 after initiation of DSS treatment. ( F ) Colon sections stained with hematoxylin and eosin on day 7 after initiation of DSS treatment. (Scale bars, 80 μm.) ( G and H ) CD4 + T cell transfer model of colitis. Naive CD4 + T cells from donor mice were transferred to recipient mice (donor > recipient). ( G ) Body weight (% relative to weight on day 0) on the indicated day after initiation of DSS treatment. C57BL/6J > Lrba +/+ Rag2 −/− ( n = 5), C57BL/6J > Lrba −/− Rag2 −/− ( n = 5). ( H ) DAI on day 16 after naive CD4 + T cell transfer. ( I , Left ) Flow cytometry of colon lamina propria cells from Lrba +/+ and Lrba −/− mice, assessing expression of CTLA4 in CD3 + CD4 + FoxP3 + cell population. ( I , Right ) Quantification of CTLA4 protein expression in colon lamina propria CD3 + CD4 + FoxP3 + cells (mean fluorescence intensity normalized to wild type). ( J – M ) Adoptive transfer of in vitro differentiated BMDCs ( J and K ) or BMpDCs ( L and M ) from donor mice to recipient mice (donor > recipient). ( J and L ) Body weight (% relative to weight on day 0) on the indicated day after initiation of DSS treatment. Lrba +/+ BMDCs > Lrba +/+ ( n = 5), Lrba −/− BMDCs > Lrba +/+ ( n = 4) for J and K . Lrba +/+ BMpDCs > Lrba +/+ ( n = 5), Lrba −/− BMpDCs > Lrba +/+ ( n = 5) for L and M . ( K and M ) DAI on day 10 after initiation of DSS treatment. ( N ) Flow cytometry of peripheral blood cells from BM chimeric mice (donor > recipient) before and after diphtheria toxin (DT) treatment (three doses, day −4, −2, and 2 relative to DSS initiation), assessing expression of CD11c and MHC II. Numbers above boxed regions represent percent cells in each. ( O ) Body weight (% relative to weight on day 0) and ( P ) DAI on day 10 after initiation of DSS treatment of BM chimeric mice (donor > recipient) treated with DT to ablate DCs. All mice were treated with DT. Each symbol ( A , B , D , E , H , I , K , M , O , and P ) represents an individual mouse. * P
Figure Legend Snippet: DCs promote DSS-induced colitis in Lrba −/− mice. ( A – F ) BM transplantation was performed using mice of the indicated genotypes (donor > recipient). ( A and C ) Body weight (% relative to weight on day 0) on day 10 ( A ) or on the indicated day ( C ) after initiation of DSS treatment. Lrba +/+ > Lrba +/+ ( n = 5), Lrba −/− > Lrba +/+ ( n = 4), Lrba +/+ > Lrba −/− ( n = 3), Lrba −/− > Lrba −/− ( n = 5) for A. Lrba +/+ Rag2 −/− > Rag2 −/− ( n = 4), Lrba −/− Rag2 −/− > Rag2 −/− ( n = 4) for C. ( B and D ) DAI and ( E ) colon length on day 10 after initiation of DSS treatment. ( F ) Colon sections stained with hematoxylin and eosin on day 7 after initiation of DSS treatment. (Scale bars, 80 μm.) ( G and H ) CD4 + T cell transfer model of colitis. Naive CD4 + T cells from donor mice were transferred to recipient mice (donor > recipient). ( G ) Body weight (% relative to weight on day 0) on the indicated day after initiation of DSS treatment. C57BL/6J > Lrba +/+ Rag2 −/− ( n = 5), C57BL/6J > Lrba −/− Rag2 −/− ( n = 5). ( H ) DAI on day 16 after naive CD4 + T cell transfer. ( I , Left ) Flow cytometry of colon lamina propria cells from Lrba +/+ and Lrba −/− mice, assessing expression of CTLA4 in CD3 + CD4 + FoxP3 + cell population. ( I , Right ) Quantification of CTLA4 protein expression in colon lamina propria CD3 + CD4 + FoxP3 + cells (mean fluorescence intensity normalized to wild type). ( J – M ) Adoptive transfer of in vitro differentiated BMDCs ( J and K ) or BMpDCs ( L and M ) from donor mice to recipient mice (donor > recipient). ( J and L ) Body weight (% relative to weight on day 0) on the indicated day after initiation of DSS treatment. Lrba +/+ BMDCs > Lrba +/+ ( n = 5), Lrba −/− BMDCs > Lrba +/+ ( n = 4) for J and K . Lrba +/+ BMpDCs > Lrba +/+ ( n = 5), Lrba −/− BMpDCs > Lrba +/+ ( n = 5) for L and M . ( K and M ) DAI on day 10 after initiation of DSS treatment. ( N ) Flow cytometry of peripheral blood cells from BM chimeric mice (donor > recipient) before and after diphtheria toxin (DT) treatment (three doses, day −4, −2, and 2 relative to DSS initiation), assessing expression of CD11c and MHC II. Numbers above boxed regions represent percent cells in each. ( O ) Body weight (% relative to weight on day 0) and ( P ) DAI on day 10 after initiation of DSS treatment of BM chimeric mice (donor > recipient) treated with DT to ablate DCs. All mice were treated with DT. Each symbol ( A , B , D , E , H , I , K , M , O , and P ) represents an individual mouse. * P

Techniques Used: Mouse Assay, Transplantation Assay, Staining, Flow Cytometry, Cytometry, Expressing, Fluorescence, Adoptive Transfer Assay, In Vitro

40) Product Images from "Thymus-derived rather than tumor-induced regulatory T cells predominate in brain tumors"

Article Title: Thymus-derived rather than tumor-induced regulatory T cells predominate in brain tumors

Journal: Neuro-Oncology

doi: 10.1093/neuonc/nor134

Brain-resident and splenic Tregs and conventional CD4 + T cells differentially express GITR, CD62L, CD103, and CD45RB in an orthotopic GL261 cell-based mouse brain tumor model at 3-weeks postoperative (WPO). (A) Representative flow cytometry plots of CD4
Figure Legend Snippet: Brain-resident and splenic Tregs and conventional CD4 + T cells differentially express GITR, CD62L, CD103, and CD45RB in an orthotopic GL261 cell-based mouse brain tumor model at 3-weeks postoperative (WPO). (A) Representative flow cytometry plots of CD4

Techniques Used: Flow Cytometry, Cytometry

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Article Snippet: .. Flow Cytometry Viable, red blood cell-depleted single splenocytes were stained with mAb (all from BD Pharmingen) against CD4 (RM4-5), CD8α (53-6.7), CD3 (500A2), CD44 (1M7) and NKp46 (29A1.4), CD69 (H1.2F3), CD103 (M290), CD62L (MEL-14). .. After washing the cells, samples were analyzed using a FortessaX20 analyzer (BD Biosciences, CA).

Article Title: HIV-1 vaccination by needle-free oral injection induces strong mucosal immunity and protects against SHIV challenge
Article Snippet: .. Rectal Biopsy samples were digested with 200 U ml− 1 Collagenase-IV (Worthington) and 0.03% DNAse-I (Life) for two hours before mechanical disruption with a syringe and needle followed by washing with RPMI supplemented with 10% FBS and stained for phenotypic markers, CD3 (BD, SP34-2, Cat# 558124, 1:200), CD4 (BD, SK3, Custom, 1:2000 dilution), CD8 (BD, RPA-T8, Custom, 1:600 dilution), CD45RA (BD, 5H9, Cat# 561216, 1:40 dilution), CCR7 (BD, 150503, Cat# 562381, 1:50 dilution), CCR5 (BD, 3A9, Cat# 742913, 1:40 dilution), HLA-DR (BD, G46-6, Custom, 1:500 dilution), and LIVE/DEAD for flow cytometry analysis. .. Rhesus macaque tissue digestion and flow cytometry Uninfected rhesus macaques scheduled for necropsy were euthanized and their buccal tissue, sublingual tissue, and submandibular, submental, and inguinal lymph nodes were collected.

Blocking Assay:

Article Title: Local Administration of GITR Agonistic Antibody Induces a Stronger Antitumor Immunity than Systemic Delivery
Article Snippet: .. Consecutive cryostat tissue sections (5 μm) were mounted on glass slides and fixed in 99.5% ethanol for 20 min. After blocking with normal rat serum, the sections were stained with rat anti-mouse CD4 and CD8 (BD Biosciences). ..

Incubation:

Article Title: Persistent Malfunction of Glymphatic and Meningeal Lymphatic Drainage in a Mouse Model of Subarachnoid Hemorrhage
Article Snippet: .. Cell suspension was incubated with antibody dye CD3 (1:400, eBioscience, Cat #145-2C11), CD4 (1:400, eBioscience Cat #17-0041-83), or CD8 (1:400, BD Bioscience Cat #335787) for 30 minutes at 4℃. .. Data were acquired on an LSR II cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using FlowJo Pro software (FlowJo, LLC, Ashland, OR, USA).

Staining:

Article Title: TIGIT and PD-1 dual checkpoint blockade enhances antitumor immunity and survival in GBM
Article Snippet: .. Immune cells were stained for surface markers using fluorescence-conjugated monoclonal antibodies including CD45 (clone HI30, BD Biosciences), CD3 (clone UCHT1, BD Biosciences), CD4 (clone L200, BD Biosciences), CD8 (clone SK1, BD Biosciences), PD1 (clone EH12.1, BD Biosciences), and TIGIT (clone 741182, R & D Systems). .. Flow cytometry was performed using FACS LSR Fortessa™ (BD Biosciences), and data was analyzed using FlowJo software (Treestar).

Article Title: BCG Vaccination Prevents Reactivation of Latent Lymphatic Murine Tuberculosis Independently of CD4+ T Cells
Article Snippet: .. Flow Cytometry Viable, red blood cell-depleted single splenocytes were stained with mAb (all from BD Pharmingen) against CD4 (RM4-5), CD8α (53-6.7), CD3 (500A2), CD44 (1M7) and NKp46 (29A1.4), CD69 (H1.2F3), CD103 (M290), CD62L (MEL-14). .. After washing the cells, samples were analyzed using a FortessaX20 analyzer (BD Biosciences, CA).

Article Title: HIV-1 vaccination by needle-free oral injection induces strong mucosal immunity and protects against SHIV challenge
Article Snippet: .. Rectal Biopsy samples were digested with 200 U ml− 1 Collagenase-IV (Worthington) and 0.03% DNAse-I (Life) for two hours before mechanical disruption with a syringe and needle followed by washing with RPMI supplemented with 10% FBS and stained for phenotypic markers, CD3 (BD, SP34-2, Cat# 558124, 1:200), CD4 (BD, SK3, Custom, 1:2000 dilution), CD8 (BD, RPA-T8, Custom, 1:600 dilution), CD45RA (BD, 5H9, Cat# 561216, 1:40 dilution), CCR7 (BD, 150503, Cat# 562381, 1:50 dilution), CCR5 (BD, 3A9, Cat# 742913, 1:40 dilution), HLA-DR (BD, G46-6, Custom, 1:500 dilution), and LIVE/DEAD for flow cytometry analysis. .. Rhesus macaque tissue digestion and flow cytometry Uninfected rhesus macaques scheduled for necropsy were euthanized and their buccal tissue, sublingual tissue, and submandibular, submental, and inguinal lymph nodes were collected.

Article Title: Mice Selected for Acute Inflammation Present Altered Immune Response during Pristane-Induced Arthritis Progression
Article Snippet: .. The cell pellets were stained with fluorophore-conjugated antibodies specific for the cell surface markers F4/80, Gr-1, CD11b, MHC class II, CD19, CD3, CD4, and CD8 (BD Biosciences) for 30 minutes at 4°C in the dark. .. After washing with FACS buffer (PBS + 1% FBS), cells were acquired by a FACSCanto II flow cytometer using BD FACSDiva software (BD Biosciences).

Article Title: Local Administration of GITR Agonistic Antibody Induces a Stronger Antitumor Immunity than Systemic Delivery
Article Snippet: .. Consecutive cryostat tissue sections (5 μm) were mounted on glass slides and fixed in 99.5% ethanol for 20 min. After blocking with normal rat serum, the sections were stained with rat anti-mouse CD4 and CD8 (BD Biosciences). ..

Article Title: In vivo monitoring of transfected DNA, gene expression kinetics, and cellular immune responses in mice immunized with a human NIS gene-expressing plasmid
Article Snippet: .. Anti-mouse CD3 antibody (3 μg/mL; BD Pharmingen) and anti-mouse CD28 antibody (3 μg/mL; BD Pharmingen) were added to the culture media to optimize restimulation., The restimulated cells were washed in PBS and fixed in 4% paraformaldehyde (PFA)–PBS for 5 min at 37°C after CD4 (BD Pharmingen) or CD8 (BD Pharmingen) staining to define the T-cell type. .. Cells (1 × 106 ) were then resuspended in 1% Saponin/5% skim-milk-PBS (S/M-PBS) and incubated with fluorescence-conjugated IL-4 (BD Pharmingen) or IFN-γ (BD Pharmingen) primary antibodies for 30 min at RT.

Recombinase Polymerase Amplification:

Article Title: HIV-1 vaccination by needle-free oral injection induces strong mucosal immunity and protects against SHIV challenge
Article Snippet: .. Rectal Biopsy samples were digested with 200 U ml− 1 Collagenase-IV (Worthington) and 0.03% DNAse-I (Life) for two hours before mechanical disruption with a syringe and needle followed by washing with RPMI supplemented with 10% FBS and stained for phenotypic markers, CD3 (BD, SP34-2, Cat# 558124, 1:200), CD4 (BD, SK3, Custom, 1:2000 dilution), CD8 (BD, RPA-T8, Custom, 1:600 dilution), CD45RA (BD, 5H9, Cat# 561216, 1:40 dilution), CCR7 (BD, 150503, Cat# 562381, 1:50 dilution), CCR5 (BD, 3A9, Cat# 742913, 1:40 dilution), HLA-DR (BD, G46-6, Custom, 1:500 dilution), and LIVE/DEAD for flow cytometry analysis. .. Rhesus macaque tissue digestion and flow cytometry Uninfected rhesus macaques scheduled for necropsy were euthanized and their buccal tissue, sublingual tissue, and submandibular, submental, and inguinal lymph nodes were collected.

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    Becton Dickinson anti cd45rb pe
    Brain-resident and splenic Tregs and conventional CD4 + T cells differentially express GITR, CD62L, CD103, and <t>CD45RB</t> in an orthotopic GL261 cell-based mouse brain tumor model at 3-weeks postoperative (WPO). (A) Representative flow cytometry plots of CD4
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    Brain-resident and splenic Tregs and conventional CD4 + T cells differentially express GITR, CD62L, CD103, and CD45RB in an orthotopic GL261 cell-based mouse brain tumor model at 3-weeks postoperative (WPO). (A) Representative flow cytometry plots of CD4

    Journal: Neuro-Oncology

    Article Title: Thymus-derived rather than tumor-induced regulatory T cells predominate in brain tumors

    doi: 10.1093/neuonc/nor134

    Figure Lengend Snippet: Brain-resident and splenic Tregs and conventional CD4 + T cells differentially express GITR, CD62L, CD103, and CD45RB in an orthotopic GL261 cell-based mouse brain tumor model at 3-weeks postoperative (WPO). (A) Representative flow cytometry plots of CD4

    Article Snippet: Cells were stained in PBS + 1% bovine serum albumin (Sigma-Aldrich) and stained with anti-CD4-PB (pacific blue) or anti-CD4-PE (phycoerythrin)-Cy7 (RM4-5; Ebioscience), anti-CD45RB-PE (16A; Becton Dickinson [BD]), anti-CD62L-FITC (fluorescein isothiocyanate) (MEL-14; BD), CD127-FITC (A7R34; Ebioscience), GITR-APC (allophycocyanin) (DTA-1; Ebioscience), and CD103-FITC (M290; BD) for 30 min on ice.

    Techniques: Flow Cytometry, Cytometry