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Nycomed peripheral blood mononuclear cells pbmc
A higher proportion of <t>CD4</t> + T cells express CD25 in patients with active systemic lupus erythematosus (SLE), although the proportion of CD25 hi remains similar to controls. Peripheral blood mononuclear cells were stained with anti-CD4-APC and an IgG1-PE isotype control antibody to establish levels of background staining. Lymphocytes were gated using forward-scatter/side-scatter characteristics. (a) Representative example of a PE-labelled isotype control and anti-CD25 staining from a control individual. (b) Staining pattern of an individual with active SLE with a particularly marked increase in the proportion of CD4 + T cells expressing CD25 (1·4%). Note also the marked reduction in the proportion of lymphocytes that are CD4 + (8.88% versus 37% in a). (c) Comparing 18 controls, 12 patients with active SLE and six with inactive disease, there was a significant increase in the percentage of CD25 + in those with active disease. P
Peripheral Blood Mononuclear Cells Pbmc, supplied by Nycomed, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Natural regulatory T cells: number and function are normal in the majority of patients with lupus nephritis"

Article Title: Natural regulatory T cells: number and function are normal in the majority of patients with lupus nephritis

Journal: Clinical and Experimental Immunology

doi: 10.1111/j.1365-2249.2008.03665.x

A higher proportion of CD4 + T cells express CD25 in patients with active systemic lupus erythematosus (SLE), although the proportion of CD25 hi remains similar to controls. Peripheral blood mononuclear cells were stained with anti-CD4-APC and an IgG1-PE isotype control antibody to establish levels of background staining. Lymphocytes were gated using forward-scatter/side-scatter characteristics. (a) Representative example of a PE-labelled isotype control and anti-CD25 staining from a control individual. (b) Staining pattern of an individual with active SLE with a particularly marked increase in the proportion of CD4 + T cells expressing CD25 (1·4%). Note also the marked reduction in the proportion of lymphocytes that are CD4 + (8.88% versus 37% in a). (c) Comparing 18 controls, 12 patients with active SLE and six with inactive disease, there was a significant increase in the percentage of CD25 + in those with active disease. P
Figure Legend Snippet: A higher proportion of CD4 + T cells express CD25 in patients with active systemic lupus erythematosus (SLE), although the proportion of CD25 hi remains similar to controls. Peripheral blood mononuclear cells were stained with anti-CD4-APC and an IgG1-PE isotype control antibody to establish levels of background staining. Lymphocytes were gated using forward-scatter/side-scatter characteristics. (a) Representative example of a PE-labelled isotype control and anti-CD25 staining from a control individual. (b) Staining pattern of an individual with active SLE with a particularly marked increase in the proportion of CD4 + T cells expressing CD25 (1·4%). Note also the marked reduction in the proportion of lymphocytes that are CD4 + (8.88% versus 37% in a). (c) Comparing 18 controls, 12 patients with active SLE and six with inactive disease, there was a significant increase in the percentage of CD25 + in those with active disease. P

Techniques Used: Staining, Expressing

Phenotypic comparison of CD25 subsets. Active lupus is associated with phenotypic changes in the CD25 – subset. Peripheral blood mononuclear cells were triple-stained for CD4, CD25 and expression of markers associated with CD4 + CD25 hi T regs or T cell activation. Subsets were gated using CD25 expression level and the proportion of each subset expressing the marker determined by comparison with staining with the isotype control antibody. Values shown are median percentage positive, with figures in brackets the 25th and 75th percentiles. Eleven controls, 11 with active systemic lupus erythematosus, five with inactive disease. (a) CD4 + T cells expressing CD25 at levels above the upper limit of CD25 expression by CD4 – lymphocytes. (b) The 2% CD4 + T cells expressing the highest CD25 levels (definition of CD25 hi used for FACS separation). (c) CD25 int . (d) CD25 – . CCR7 from 10 controls, nine patients with active and five with inactive disease. ×: P -value comparing controls and patients with inactive lupus; ♦, P -value comparing controls and patients with active lupus, †: P -value comparing patients with active and inactive lupus. Statistical comparison was made using the Kruskal–Wallis test with Dunn's post-test. FACS, fluorescence activated cell sorter; HLA-DR, human leucocyte antigen D-related.
Figure Legend Snippet: Phenotypic comparison of CD25 subsets. Active lupus is associated with phenotypic changes in the CD25 – subset. Peripheral blood mononuclear cells were triple-stained for CD4, CD25 and expression of markers associated with CD4 + CD25 hi T regs or T cell activation. Subsets were gated using CD25 expression level and the proportion of each subset expressing the marker determined by comparison with staining with the isotype control antibody. Values shown are median percentage positive, with figures in brackets the 25th and 75th percentiles. Eleven controls, 11 with active systemic lupus erythematosus, five with inactive disease. (a) CD4 + T cells expressing CD25 at levels above the upper limit of CD25 expression by CD4 – lymphocytes. (b) The 2% CD4 + T cells expressing the highest CD25 levels (definition of CD25 hi used for FACS separation). (c) CD25 int . (d) CD25 – . CCR7 from 10 controls, nine patients with active and five with inactive disease. ×: P -value comparing controls and patients with inactive lupus; ♦, P -value comparing controls and patients with active lupus, †: P -value comparing patients with active and inactive lupus. Statistical comparison was made using the Kruskal–Wallis test with Dunn's post-test. FACS, fluorescence activated cell sorter; HLA-DR, human leucocyte antigen D-related.

Techniques Used: Staining, Expressing, Activation Assay, Marker, FACS, Fluorescence

2) Product Images from "NKG2D expression in CD4+ T lymphocytes as a marker of senescence in the aged immune system"

Article Title: NKG2D expression in CD4+ T lymphocytes as a marker of senescence in the aged immune system

Journal: Age

doi: 10.1007/s11357-010-9200-6

CD69 expression in response to CMV antigens and to the influenza vaccine. PBMCs from elderly individuals were stimulated for 18 h and CD69 expression was evaluated by flow cytometry. a Representative dot plots of the frequency of CMV- and influenza vaccine-specific CD4 T cells with and without NKG2D expression. Percentages of positive cells in this representative experiment are expressed in dot plots. b Histograms summarize the percentage of CD69-cells positive for CMV and influenza vaccine in the CD4+ CD28 null NKG2D+ ( black bars ) and CD4+ CD28 null NKG2D- subsets ( white bars ) obtained from 11 elderly donors. Paired t test was used to compare paired frequencies
Figure Legend Snippet: CD69 expression in response to CMV antigens and to the influenza vaccine. PBMCs from elderly individuals were stimulated for 18 h and CD69 expression was evaluated by flow cytometry. a Representative dot plots of the frequency of CMV- and influenza vaccine-specific CD4 T cells with and without NKG2D expression. Percentages of positive cells in this representative experiment are expressed in dot plots. b Histograms summarize the percentage of CD69-cells positive for CMV and influenza vaccine in the CD4+ CD28 null NKG2D+ ( black bars ) and CD4+ CD28 null NKG2D- subsets ( white bars ) obtained from 11 elderly donors. Paired t test was used to compare paired frequencies

Techniques Used: Expressing, Flow Cytometry, Cytometry

Differentiated status of CD4+ subsets defined by IL-2/IFN-γ production and TREC content. a PBMCs were stimulated for 6 h with soluble anti-CD3 (1 μg/mL). The responder cells were analyzed for intracellular IL-2-APC staining in the indicated populations. b Cells were treated as described previously and IFN-γ expression was analyzed by intracellular staining. Percentages of positive cells in the indicated populations in this representative experiment are expressed on the histogram-plots. Histograms summarize the percentage of IFN-γ-positive cells in CD4+ CD28 null and in CD4+ CD28+ lymphocytes NKG2D− and NKG2D + (mean ± SEM) from the six elderly donors tested. A paired t test was used to compare paired means. c Dose-response curves of the IFN-γ production by CD4 subsets defined by CD28 and NKG2D expression in response to anti-CD3. The cells were cultured for 6 h in medium alone or with increasing concentrations of anti-CD3 (1–1,000 ng/mL). A fluorescence analysis was carried out as previously described. One representative experiment of three is shown. d Quantification of TREC copy number. CD28+, CD28 null NKG2D − , and CD28 null NKG2D+ populations were isolated by sorting (magnetic bead separation) and the TREC copy number was determined by real-time PCR. Experiments were conducted in duplicate and bars represented results from three aging donors (mean ± SEM)
Figure Legend Snippet: Differentiated status of CD4+ subsets defined by IL-2/IFN-γ production and TREC content. a PBMCs were stimulated for 6 h with soluble anti-CD3 (1 μg/mL). The responder cells were analyzed for intracellular IL-2-APC staining in the indicated populations. b Cells were treated as described previously and IFN-γ expression was analyzed by intracellular staining. Percentages of positive cells in the indicated populations in this representative experiment are expressed on the histogram-plots. Histograms summarize the percentage of IFN-γ-positive cells in CD4+ CD28 null and in CD4+ CD28+ lymphocytes NKG2D− and NKG2D + (mean ± SEM) from the six elderly donors tested. A paired t test was used to compare paired means. c Dose-response curves of the IFN-γ production by CD4 subsets defined by CD28 and NKG2D expression in response to anti-CD3. The cells were cultured for 6 h in medium alone or with increasing concentrations of anti-CD3 (1–1,000 ng/mL). A fluorescence analysis was carried out as previously described. One representative experiment of three is shown. d Quantification of TREC copy number. CD28+, CD28 null NKG2D − , and CD28 null NKG2D+ populations were isolated by sorting (magnetic bead separation) and the TREC copy number was determined by real-time PCR. Experiments were conducted in duplicate and bars represented results from three aging donors (mean ± SEM)

Techniques Used: Staining, Expressing, Cell Culture, Fluorescence, Isolation, Real-time Polymerase Chain Reaction

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Article Title: Reassessing the role of HLA-DRB3 T cell responses: Evidence for significant expression and complementary antigen presentation
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Article Title: Identification of Immunodominant Epitopes Derived from the Respiratory Syncytial Virus Fusion Protein That Are Recognized by Human CD4 T Cells
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Centrifugation:

Article Title: Reassessing the role of HLA-DRB3 T cell responses: Evidence for significant expression and complementary antigen presentation
Article Snippet: .. For peptide stimulations, PBMC were isolated from the heparinized blood of immunized healthy donors by density centrifugation (Lymphoprep; Nycomed Pharma AS Diagnostics, Oslo, Norway). .. When T CD4+ cells were stimulated, these were isolated from PBMC using a negative selection method (CD4+ T Cell Isolation Kit II, Miltenyi Biotech, Auburn, CA) following manufacturer instructions and using the AutoMacs system (Miltenyi Biotech, Auburn, CA) to perform the separation.

Article Title: Suppression of autologous peripheral blood mononuclear cell proliferation by alveolar macrophages from young infants
Article Snippet: BALF cells were washed twice and re-suspended at a concentration of 1 × 106 cells/ml in RPMI-CS and kept on ice. .. PBMC were isolated by separation on a density gradient using Lymphoprep (Nycomed, Oslo, Norway) at 20°C by centrifugation at 800 g for 20 min. PBMC were washed twice in RPMI-CS and then re-suspended at 5 × 105 cells/ml. .. To determine the BALF and PBMC cell differential count, 1 × 104 unfixed cells were cytocentrifuged at 800 rpm for 5 min onto a microscope slide (Shandon Scientific, Runcorn, Cheshire, UK), air-dried and stained with Diff-Quik (Dade Behring, Deerfield, IL, USA).

Article Title: Identification of Immunodominant Epitopes Derived from the Respiratory Syncytial Virus Fusion Protein That Are Recognized by Human CD4 T Cells
Article Snippet: .. PBMC were isolated by density centrifugation with Lymphoprep (Nycomed Pharma). .. PBMC were either thawed from cryopreserved samples (−135°C) in RPMI 1640 (Gibco)-10% dimethyl sulfoxide-30% fetal bovine serum (HyClone), or used fresh.

Article Title: Aspirin down-regulates tryptophan degradation in stimulated human peripheral blood mononuclear cells in vitro
Article Snippet: .. PBMC were isolated from whole blood obtained from healthy voluntary blood donors by density centrifugation (Lymphoprep, Nycomed Pharma AS, Oslo, Norway). .. Cells were maintained in RPMI-1640 (PAA-Laboratories, Linz, Austria) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Biochrom, Berlin, Germany), 2 mM l -glutamine (Serva, Heidelberg, Germany) and 50 µg/ml gentamycin (Bio-Whitaker, Walkersville, MD, USA).

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    Nycomed peripheral blood mononuclear cells pbmc
    Dose–response curves for <t>ADCC.</t> The procedures for OxLDL incubated and Ab treatment were the same as described in the legend for Fig. . As effectors normal rat <t>PBMC</t> were added to the culture instead of complement. Note increased 51Cr release
    Peripheral Blood Mononuclear Cells Pbmc, supplied by Nycomed, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/peripheral blood mononuclear cells pbmc/product/Nycomed
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Nycomed treg peripheral blood mononuclear cells pbmcs
    TNFR2-agonist plus rapamycin expanded <t>Treg</t> inhibit inflammation in a humanised mouse model. (A) Schematic overview of the humanised skin inflammation mouse model used. In brief, SCID mice were transplanted with a human skin graft, 21 days after engraftment, PBS (as a control), or allogeneic human <t>PBMC</t> (huPBMC) only or huPBMC plus Treg of interest (at a ratio of 1:1) were injected intra peritoneally. 26 days later the animals were sacrificed to analyze the mouse spleen and human skin grafts. Rap Treg and R/T Treg refer to low purity MACS-isolated Treg expanded for 16-days in the presence of rapamycin-only or TNFR2-agonist plus rapamycin, respectively. (B) Representative photographs of spleens derived from mice infused with PBS, huPBMC only, or huPBMC plus Treg of interest, 21 days after the skin transplantation. Cumulative data showing the weight of spleens derived from N = 4 mice (right panel; n.d. = not determined). (C) Representative photographs showing histology (HE staining) of human skin grafts. Left panel: 10 x magnification. Right panel shows the epidermal thickness (μm) of human skin grafts following infusion of PBS, huPBMC, huPBMC plus Rap Treg or huPBMC plus R/T Treg. Mean ± SD, N = 6. (D) Immunohistochemistry of human CD3+ (brown) T cell infiltration in the human dermis. A representative photograph of N = 4 as presented in the cumulative data graph is shown (right panel; Mean ± SD). 20 x magnification. Wilcoxon paried t Test was used to compare the group of mice infused with huPBMC only with other groups of mice infused with huPBMC plus Treg of interest. Asterisks indicate significant differences.
    Treg Peripheral Blood Mononuclear Cells Pbmcs, supplied by Nycomed, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/treg peripheral blood mononuclear cells pbmcs/product/Nycomed
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Dose–response curves for ADCC. The procedures for OxLDL incubated and Ab treatment were the same as described in the legend for Fig. . As effectors normal rat PBMC were added to the culture instead of complement. Note increased 51Cr release

    Journal: Cell Stress & Chaperones

    Article Title: Evidence of a role for both anti-Hsp70 antibody and endothelial surface membrane Hsp70 in atherosclerosis

    doi: 10.1007/s12192-013-0404-4

    Figure Lengend Snippet: Dose–response curves for ADCC. The procedures for OxLDL incubated and Ab treatment were the same as described in the legend for Fig. . As effectors normal rat PBMC were added to the culture instead of complement. Note increased 51Cr release

    Article Snippet: To determine antibody-dependent cellular cytotoxicity (ADCC), peripheral blood mononuclear cells (PBMC) were isolated from healthy rats by density centrifugation (Lymphoprep, density 1,083; Nycomed Pharmaceuticals Oslo, Norway) as described previously (Jurgens et al. ).

    Techniques: Incubation

    CD69 expression and proliferative capacity of CD4+ and CD8+ depending on the functional capacity. Whole blood from the BI groups of elders (group 0 + 1, n = 12 and group 2 + 3, n = 14) was stimulated for 18 h and expression of CD69 in CD4+ and CD8+ T cells was evaluated by flow cytometry. Proliferative capacity of CD4+ and CD8+ T cells subsets was also evaluated in the two groups (group 0 + 1, n = 19 and group 2 + 3, n = 16) by labeling the PBMC with CFSE. Cells were stained and 1 × 10 5 cells were acquired per experiment. a Representative dot plots showing the frequency of CD69 expression in CD4+ and CD8+ subset from elderly with different functional status. Cells were stimulated using anti-CD3 (10 ng/mL). Percentage of positive cells in each subpopulation in this representative experiment is expressed in the upper right corner and summarized results from all donors (median and IR) were also expressed in dot plots. b Proliferative capacity of CD4+ and CD8+ T cells subsets in response to anti-CD3. PBMC were labeled with CFSE (1.5 μM) and cultured in presence of anti-CD3 (10 ng/mL) for 5 days. Percentage of dividing CD4+ and CD8+ T cells is represented. Bars represent results from the grouped elders (mean ± SEM). c Expression of CD69 into de CD4+ and CD8+ T cell subset was analyzed in the same way as in Fig. 5a in response to a CMV supernatant (10 4 PFU/mL). d Proliferative capacity of CD4+ and CD8+ T-cell subsets in response to the CMV supernatant. Bars represent resulted from the grouped elders (mean ± SEM). The Student’s t test (when data were normally distributed) and Mann–Whitney non-parametric (when data were not normally distributed) methods were used to compare frequencies between groups. p values are depicted in the panels

    Journal: Age

    Article Title: Relationship between functional ability in older people, immune system status, and intensity of response to CMV

    doi: 10.1007/s11357-011-9240-6

    Figure Lengend Snippet: CD69 expression and proliferative capacity of CD4+ and CD8+ depending on the functional capacity. Whole blood from the BI groups of elders (group 0 + 1, n = 12 and group 2 + 3, n = 14) was stimulated for 18 h and expression of CD69 in CD4+ and CD8+ T cells was evaluated by flow cytometry. Proliferative capacity of CD4+ and CD8+ T cells subsets was also evaluated in the two groups (group 0 + 1, n = 19 and group 2 + 3, n = 16) by labeling the PBMC with CFSE. Cells were stained and 1 × 10 5 cells were acquired per experiment. a Representative dot plots showing the frequency of CD69 expression in CD4+ and CD8+ subset from elderly with different functional status. Cells were stimulated using anti-CD3 (10 ng/mL). Percentage of positive cells in each subpopulation in this representative experiment is expressed in the upper right corner and summarized results from all donors (median and IR) were also expressed in dot plots. b Proliferative capacity of CD4+ and CD8+ T cells subsets in response to anti-CD3. PBMC were labeled with CFSE (1.5 μM) and cultured in presence of anti-CD3 (10 ng/mL) for 5 days. Percentage of dividing CD4+ and CD8+ T cells is represented. Bars represent results from the grouped elders (mean ± SEM). c Expression of CD69 into de CD4+ and CD8+ T cell subset was analyzed in the same way as in Fig. 5a in response to a CMV supernatant (10 4 PFU/mL). d Proliferative capacity of CD4+ and CD8+ T-cell subsets in response to the CMV supernatant. Bars represent resulted from the grouped elders (mean ± SEM). The Student’s t test (when data were normally distributed) and Mann–Whitney non-parametric (when data were not normally distributed) methods were used to compare frequencies between groups. p values are depicted in the panels

    Article Snippet: To analyze the proliferation status of CD4+ and CD8+ T cells, peripheral blood mononuclear cells (PBMC) were isolated by centrifugation on Ficoll-Hypaque gradients (Lymphoprep; Nycomed, Oslo, Norway).

    Techniques: Expressing, Functional Assay, Flow Cytometry, Cytometry, Labeling, Staining, Cell Culture, MANN-WHITNEY

    CD69 expression in response to CMV antigens and to the influenza vaccine. PBMCs from elderly individuals were stimulated for 18 h and CD69 expression was evaluated by flow cytometry. a Representative dot plots of the frequency of CMV- and influenza vaccine-specific CD4 T cells with and without NKG2D expression. Percentages of positive cells in this representative experiment are expressed in dot plots. b Histograms summarize the percentage of CD69-cells positive for CMV and influenza vaccine in the CD4+ CD28 null NKG2D+ ( black bars ) and CD4+ CD28 null NKG2D- subsets ( white bars ) obtained from 11 elderly donors. Paired t test was used to compare paired frequencies

    Journal: Age

    Article Title: NKG2D expression in CD4+ T lymphocytes as a marker of senescence in the aged immune system

    doi: 10.1007/s11357-010-9200-6

    Figure Lengend Snippet: CD69 expression in response to CMV antigens and to the influenza vaccine. PBMCs from elderly individuals were stimulated for 18 h and CD69 expression was evaluated by flow cytometry. a Representative dot plots of the frequency of CMV- and influenza vaccine-specific CD4 T cells with and without NKG2D expression. Percentages of positive cells in this representative experiment are expressed in dot plots. b Histograms summarize the percentage of CD69-cells positive for CMV and influenza vaccine in the CD4+ CD28 null NKG2D+ ( black bars ) and CD4+ CD28 null NKG2D- subsets ( white bars ) obtained from 11 elderly donors. Paired t test was used to compare paired frequencies

    Article Snippet: To analyze the differentiation status of CD4+ T cells, peripheral blood mononuclear cells (PBMC) were isolated by centrifugation on Ficoll–Hypaque gradients (Lymphoprep; Nycomed, Oslo, Norway).

    Techniques: Expressing, Flow Cytometry, Cytometry

    Differentiated status of CD4+ subsets defined by IL-2/IFN-γ production and TREC content. a PBMCs were stimulated for 6 h with soluble anti-CD3 (1 μg/mL). The responder cells were analyzed for intracellular IL-2-APC staining in the indicated populations. b Cells were treated as described previously and IFN-γ expression was analyzed by intracellular staining. Percentages of positive cells in the indicated populations in this representative experiment are expressed on the histogram-plots. Histograms summarize the percentage of IFN-γ-positive cells in CD4+ CD28 null and in CD4+ CD28+ lymphocytes NKG2D− and NKG2D + (mean ± SEM) from the six elderly donors tested. A paired t test was used to compare paired means. c Dose-response curves of the IFN-γ production by CD4 subsets defined by CD28 and NKG2D expression in response to anti-CD3. The cells were cultured for 6 h in medium alone or with increasing concentrations of anti-CD3 (1–1,000 ng/mL). A fluorescence analysis was carried out as previously described. One representative experiment of three is shown. d Quantification of TREC copy number. CD28+, CD28 null NKG2D − , and CD28 null NKG2D+ populations were isolated by sorting (magnetic bead separation) and the TREC copy number was determined by real-time PCR. Experiments were conducted in duplicate and bars represented results from three aging donors (mean ± SEM)

    Journal: Age

    Article Title: NKG2D expression in CD4+ T lymphocytes as a marker of senescence in the aged immune system

    doi: 10.1007/s11357-010-9200-6

    Figure Lengend Snippet: Differentiated status of CD4+ subsets defined by IL-2/IFN-γ production and TREC content. a PBMCs were stimulated for 6 h with soluble anti-CD3 (1 μg/mL). The responder cells were analyzed for intracellular IL-2-APC staining in the indicated populations. b Cells were treated as described previously and IFN-γ expression was analyzed by intracellular staining. Percentages of positive cells in the indicated populations in this representative experiment are expressed on the histogram-plots. Histograms summarize the percentage of IFN-γ-positive cells in CD4+ CD28 null and in CD4+ CD28+ lymphocytes NKG2D− and NKG2D + (mean ± SEM) from the six elderly donors tested. A paired t test was used to compare paired means. c Dose-response curves of the IFN-γ production by CD4 subsets defined by CD28 and NKG2D expression in response to anti-CD3. The cells were cultured for 6 h in medium alone or with increasing concentrations of anti-CD3 (1–1,000 ng/mL). A fluorescence analysis was carried out as previously described. One representative experiment of three is shown. d Quantification of TREC copy number. CD28+, CD28 null NKG2D − , and CD28 null NKG2D+ populations were isolated by sorting (magnetic bead separation) and the TREC copy number was determined by real-time PCR. Experiments were conducted in duplicate and bars represented results from three aging donors (mean ± SEM)

    Article Snippet: To analyze the differentiation status of CD4+ T cells, peripheral blood mononuclear cells (PBMC) were isolated by centrifugation on Ficoll–Hypaque gradients (Lymphoprep; Nycomed, Oslo, Norway).

    Techniques: Staining, Expressing, Cell Culture, Fluorescence, Isolation, Real-time Polymerase Chain Reaction

    TNFR2-agonist plus rapamycin expanded Treg inhibit inflammation in a humanised mouse model. (A) Schematic overview of the humanised skin inflammation mouse model used. In brief, SCID mice were transplanted with a human skin graft, 21 days after engraftment, PBS (as a control), or allogeneic human PBMC (huPBMC) only or huPBMC plus Treg of interest (at a ratio of 1:1) were injected intra peritoneally. 26 days later the animals were sacrificed to analyze the mouse spleen and human skin grafts. Rap Treg and R/T Treg refer to low purity MACS-isolated Treg expanded for 16-days in the presence of rapamycin-only or TNFR2-agonist plus rapamycin, respectively. (B) Representative photographs of spleens derived from mice infused with PBS, huPBMC only, or huPBMC plus Treg of interest, 21 days after the skin transplantation. Cumulative data showing the weight of spleens derived from N = 4 mice (right panel; n.d. = not determined). (C) Representative photographs showing histology (HE staining) of human skin grafts. Left panel: 10 x magnification. Right panel shows the epidermal thickness (μm) of human skin grafts following infusion of PBS, huPBMC, huPBMC plus Rap Treg or huPBMC plus R/T Treg. Mean ± SD, N = 6. (D) Immunohistochemistry of human CD3+ (brown) T cell infiltration in the human dermis. A representative photograph of N = 4 as presented in the cumulative data graph is shown (right panel; Mean ± SD). 20 x magnification. Wilcoxon paried t Test was used to compare the group of mice infused with huPBMC only with other groups of mice infused with huPBMC plus Treg of interest. Asterisks indicate significant differences.

    Journal: PLoS ONE

    Article Title: A TNFR2-Agonist Facilitates High Purity Expansion of Human Low Purity Treg Cells

    doi: 10.1371/journal.pone.0156311

    Figure Lengend Snippet: TNFR2-agonist plus rapamycin expanded Treg inhibit inflammation in a humanised mouse model. (A) Schematic overview of the humanised skin inflammation mouse model used. In brief, SCID mice were transplanted with a human skin graft, 21 days after engraftment, PBS (as a control), or allogeneic human PBMC (huPBMC) only or huPBMC plus Treg of interest (at a ratio of 1:1) were injected intra peritoneally. 26 days later the animals were sacrificed to analyze the mouse spleen and human skin grafts. Rap Treg and R/T Treg refer to low purity MACS-isolated Treg expanded for 16-days in the presence of rapamycin-only or TNFR2-agonist plus rapamycin, respectively. (B) Representative photographs of spleens derived from mice infused with PBS, huPBMC only, or huPBMC plus Treg of interest, 21 days after the skin transplantation. Cumulative data showing the weight of spleens derived from N = 4 mice (right panel; n.d. = not determined). (C) Representative photographs showing histology (HE staining) of human skin grafts. Left panel: 10 x magnification. Right panel shows the epidermal thickness (μm) of human skin grafts following infusion of PBS, huPBMC, huPBMC plus Rap Treg or huPBMC plus R/T Treg. Mean ± SD, N = 6. (D) Immunohistochemistry of human CD3+ (brown) T cell infiltration in the human dermis. A representative photograph of N = 4 as presented in the cumulative data graph is shown (right panel; Mean ± SD). 20 x magnification. Wilcoxon paried t Test was used to compare the group of mice infused with huPBMC only with other groups of mice infused with huPBMC plus Treg of interest. Asterisks indicate significant differences.

    Article Snippet: Isolation of Treg Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation (Lymphoprep, Nycomed Pharma AS, Oslo, Norway) of buffy coats obtained from healthy blood donors (Sanquin Blood Bank, Region South-East, Netherlands) upon written informed consent, according to the Dutch law.

    Techniques: Mouse Assay, Injection, Magnetic Cell Separation, Isolation, Derivative Assay, Transplantation Assay, Staining, Immunohistochemistry