pe-anti-cd27 Search Results


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  • 89
    Immunotec pe anti cd27
    Pe Anti Cd27, supplied by Immunotec, used in various techniques. Bioz Stars score: 89/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies pe anti cd27
    Immunophenotyping of CARMIL2-deficient T cells. (A) Representative plot of viable PBMCs from one control and one patient, gated as shown then stained with <t>CD45RO/CD27</t> to determine T cell subtype population numbers. Plots show CD4 and CD8 T cells that are naïve (T N , CD45RO − CD27 + ), central memory (T CM , CD45RO + CD27 + ), effector memory (T EM , CD45RO + CD27 − ), and effector (T Eff , CD45RO − CD27 − ). Corresponding percentages are indicated in each quadrant. (B) Summary of CD45RO/CD27 staining data from CARMIL2 patients ( n = 7), along with healthy controls ( n = 6), separated based on CD4/CD8 subtype. Asterisks indicate significance levels (* p
    Pe Anti Cd27, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    BioLegend pe anti cd27
    Expansion of memory-like NK cells in BCG-vaccinated mice depends on IL-21 C57BL/6 mice were treated with PBS or immunized subcutaneously with 10 6 CFU of BCG in 100 µl of PBS. One month after vaccination, spleen, and peripheral lymph node cells were pooled. (A) CD3- NK cells were isolated and cultured with peritoneal macrophages, with or without Ag85. (B) Pooled cells were cultured with or without Ag85, in the presence of isotype-matched control antibodies or antibodies to IL-4, IL-7, IL-17, or IL-21. (C) Pooled cells from one month BCG vaccinated C57BL/6 mice were transfected with either IL-21 or scrambled siRNA (control siRNA) and cultured with or without Ag85. (D) Pooled cells from one month BCG vaccinated IL-21 knockout and respective control mice were cultured with or without Ag85. (E) Pooled cells from one month BCG vaccinated IL-21R knockout and respective control mice were cultured with or without Ag85. In all panels, after five days, expansion of <t>CD3-NKp46+CD27+</t> cells were measured by flow cytometry. Mean values and SEs are shown. Data are representative oftwo independent experiments.
    Pe Anti Cd27, supplied by BioLegend, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson pe anti cd27
    CD81 engagement induces the preferential expansion of naïve B lymphocytes. Purified naive <t>(CD27</t> - ) or memory (CD27 + ) B cells were incubated with complete medium (Control), MG81 and N81 mAbs (5 μg/ml each) (CD81), or SAC (1:30,000, vol/vol).
    Pe Anti Cd27, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 223 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beckman Coulter pe anti cd27
    IgM + IgD + <t>CD27</t> + B cells from blood and spleen share B cell clones with an identical V3-15 CDR3 during a T-independent response Amplification of V3-15-Cμ mRNA sequences was performed from naive and IgM + IgD + CD27 + B cells of a 9-year old child undergoing splenectomy and immunized against Streptococcus pneumoniae and Neisseria meningitidis (plain polysaccharidic vaccines). The following samples were analyzed: blood before immunization, blood at the time of splenectomy (i.e. 8 days after immunization), spleen, blood 5 weeks after immunization. The first V3-15-specific PCR products were further amplified with V3-15-specific FR3 and C7μ primers, and the resulting products fractionated by denaturing gel electrophoresis. A specific CDR3 size was excised from the gel after silver staining and reamplified with the same FR3 and Cμ primers, and sequences determined after cloning. Several PCR amplifications were performed from two independent cDNAs for each cell sample. The recurrent CDR3s encompassing the V3-15 and J H 3 junctions observed in the various IgM + IgD + CD27 + fractions are shown, with the most frequently occurring sequence taken as reference (CDR3 is defined as amino acids included between the conserved Cys residue of FR3 and Trp residue of J H segments). The asterisk (*) marks clones found repeatedly in independent PCR amplifications.
    Pe Anti Cd27, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 89/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pe anti cd27
    Infiltration of B cells and iNOS-expressing PCs in H. pylori + patients. ( A ) Representative gating strategy for analysis of LPLs by flow cytometry: 1) gating on lymphocytes by forward (FSC) and side scatter (SSC) properties; 2) exclusion of T cells (CD3 + ) and monocytes (CD14 + ) and gating on CD19 + B cells; 3) detection of PCs (CD20 − <t>CD27</t> ++ ), mBCs (CD20 + CD27 + ), and naive B cells (CD20 + CD27 − ); 4) detection of iNOS + CD38 ++ PCs; and 5) detection of IgA + PCs within iNOS + CD38 ++ PCs. ( B ) Quantitative analysis of the flow cytometric data of H. pylori –infected patients according to the gating strategy. Values are depicted as median plus interquartile ranges (Mann–Whitney U test).
    Pe Anti Cd27, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher pe anti cd27 mab
    somatic hypermutation (SHM) results on the VH3-23 region of IgM on <t>CD19+CD27+</t> isolated B cells. ( A ) Frequency of mutations on IgM+CD27+ B cells purified from controls and patients. Dots represent results for each subject. Controls: Black dots, all 10 clones mutated and white dots, 9/10 clones mutated. Patients: P1, * and P2, #; ( B ) Nucleotide substitution pattern for P1 (1st evaluation), P2, and control. The same pattern was observed in P1 in the 2nd evaluation. ( C ) The numbers of mutated clones from all studied clones is shown, as well as the frequency of mutations.
    Pe Anti Cd27 Mab, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson phycoerythrin pe anti cd27 mab
    CD81 engagement induces the preferential expansion of naïve B lymphocytes. Purified naive <t>(CD27</t> - ) or memory (CD27 + ) B cells were incubated with complete medium (Control), MG81 and N81 mAbs (5 μg/ml each) (CD81), or SAC (1:30,000, vol/vol).
    Phycoerythrin Pe Anti Cd27 Mab, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Pharmingen anti cd27 pe
    EBV-infected cells in the periphery of AIM patients are <t>CD27</t> positive. PBMCs were stained for expression of CD20, a pan-B-cell marker, and CD27, a memory B-cell marker (upper panel). The naive and memory B cells were then sorted by FACS and reanalyzed for purity (middle panels). The purified populations were then tested for the presence of the virus by limiting dilution DNA PCR. In this technique, serial dilutions of each population are prepared and then multiple aliquots of each dilution are tested for the virus by DNA PCR. The PCR products are separated on a gel and detected by Southern blotting.
    Anti Cd27 Pe, supplied by Pharmingen, used in various techniques. Bioz Stars score: 91/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson pe cy7 anti cd27
    EBV-infected cells in the periphery of AIM patients are <t>CD27</t> positive. PBMCs were stained for expression of CD20, a pan-B-cell marker, and CD27, a memory B-cell marker (upper panel). The naive and memory B cells were then sorted by FACS and reanalyzed for purity (middle panels). The purified populations were then tested for the presence of the virus by limiting dilution DNA PCR. In this technique, serial dilutions of each population are prepared and then multiple aliquots of each dilution are tested for the virus by DNA PCR. The PCR products are separated on a gel and detected by Southern blotting.
    Pe Cy7 Anti Cd27, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pe cy7 anti cd27
    EBV-infected cells in the periphery of AIM patients are <t>CD27</t> positive. PBMCs were stained for expression of CD20, a pan-B-cell marker, and CD27, a memory B-cell marker (upper panel). The naive and memory B cells were then sorted by FACS and reanalyzed for purity (middle panels). The purified populations were then tested for the presence of the virus by limiting dilution DNA PCR. In this technique, serial dilutions of each population are prepared and then multiple aliquots of each dilution are tested for the virus by DNA PCR. The PCR products are separated on a gel and detected by Southern blotting.
    Pe Cy7 Anti Cd27, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Miltenyi Biotec anti cd27 pe
    Gene array comparisons of CD21 + and CD21 –/low <t>CD27</t> - B cells from pSS patients using the Illumina Human Whole Genome Array
    Anti Cd27 Pe, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pharmingen anti cd27 phycoerythrin pe
    HIV-1 infected patients present with expanded populations of blood <t>CD27</t> − IgA + and CD27 − IgG + B-cells and show inverse patterns of SHM. The percentage of CD27 − IgA + (A) and CD27 − IgG + (B) among total IgA/G expressing B-cells is significantly expanded in HIV-1 infected patients (right panel) as compared with healthy controls (left panel). (C) The number of somatic hypermutations in the VH region of mRNA transcripts of CD27 − B cells (left panel) is increased in HIV-1 infected patients (black bars), as compared with healthy controls (white bars), while an opposite trend is shown for CD27 + B cells (right panel), where the number of somatic hypermutations in the VH region of mRNA transcripts is decreased in HIV-1 infected patients (black bars) as compared with healthy controls (white bars). The number below the bars indicates the number of clones analysed.
    Anti Cd27 Phycoerythrin Pe, supplied by Pharmingen, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Holzel Diagnostika anti cd27 pe antibody
    HIV-1 infected patients present with expanded populations of blood <t>CD27</t> − IgA + and CD27 − IgG + B-cells and show inverse patterns of SHM. The percentage of CD27 − IgA + (A) and CD27 − IgG + (B) among total IgA/G expressing B-cells is significantly expanded in HIV-1 infected patients (right panel) as compared with healthy controls (left panel). (C) The number of somatic hypermutations in the VH region of mRNA transcripts of CD27 − B cells (left panel) is increased in HIV-1 infected patients (black bars), as compared with healthy controls (white bars), while an opposite trend is shown for CD27 + B cells (right panel), where the number of somatic hypermutations in the VH region of mRNA transcripts is decreased in HIV-1 infected patients (black bars) as compared with healthy controls (white bars). The number below the bars indicates the number of clones analysed.
    Anti Cd27 Pe Antibody, supplied by Holzel Diagnostika, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson pe conjugated anti cd27
    The percentages of IgG + IgA + <t>CD27</t> + switch memory B cells increase after vaccination in young but not in elderly subjects
    Pe Conjugated Anti Cd27, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beckman Coulter anti cd27 pe cy5
    The percentages of IgG + IgA + <t>CD27</t> + switch memory B cells increase after vaccination in young but not in elderly subjects
    Anti Cd27 Pe Cy5, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 88/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Immunophenotyping of CARMIL2-deficient T cells. (A) Representative plot of viable PBMCs from one control and one patient, gated as shown then stained with CD45RO/CD27 to determine T cell subtype population numbers. Plots show CD4 and CD8 T cells that are naïve (T N , CD45RO − CD27 + ), central memory (T CM , CD45RO + CD27 + ), effector memory (T EM , CD45RO + CD27 − ), and effector (T Eff , CD45RO − CD27 − ). Corresponding percentages are indicated in each quadrant. (B) Summary of CD45RO/CD27 staining data from CARMIL2 patients ( n = 7), along with healthy controls ( n = 6), separated based on CD4/CD8 subtype. Asterisks indicate significance levels (* p

    Journal: Frontiers in Immunology

    Article Title: Novel CARMIL2 Mutations in Patients with Variable Clinical Dermatitis, Infections, and Combined Immunodeficiency

    doi: 10.3389/fimmu.2018.00203

    Figure Lengend Snippet: Immunophenotyping of CARMIL2-deficient T cells. (A) Representative plot of viable PBMCs from one control and one patient, gated as shown then stained with CD45RO/CD27 to determine T cell subtype population numbers. Plots show CD4 and CD8 T cells that are naïve (T N , CD45RO − CD27 + ), central memory (T CM , CD45RO + CD27 + ), effector memory (T EM , CD45RO + CD27 − ), and effector (T Eff , CD45RO − CD27 − ). Corresponding percentages are indicated in each quadrant. (B) Summary of CD45RO/CD27 staining data from CARMIL2 patients ( n = 7), along with healthy controls ( n = 6), separated based on CD4/CD8 subtype. Asterisks indicate significance levels (* p

    Article Snippet: Antibodies used were AmCyan-anti-CD3, APC-anti-CD4, PerCP-Cy5.5-anti-CD4, Pacific Blue-anti-CD8, PE-Cy7-anti-CD8 (all BD Biosciences), FITC-anti-CD45RO, PE-anti-CD27 (DAKO), and APC-anti-CD25, PE-Cy7-anti-CD127 (eBioscience).

    Techniques: Staining

    Expansion of memory-like NK cells in BCG-vaccinated mice depends on IL-21 C57BL/6 mice were treated with PBS or immunized subcutaneously with 10 6 CFU of BCG in 100 µl of PBS. One month after vaccination, spleen, and peripheral lymph node cells were pooled. (A) CD3- NK cells were isolated and cultured with peritoneal macrophages, with or without Ag85. (B) Pooled cells were cultured with or without Ag85, in the presence of isotype-matched control antibodies or antibodies to IL-4, IL-7, IL-17, or IL-21. (C) Pooled cells from one month BCG vaccinated C57BL/6 mice were transfected with either IL-21 or scrambled siRNA (control siRNA) and cultured with or without Ag85. (D) Pooled cells from one month BCG vaccinated IL-21 knockout and respective control mice were cultured with or without Ag85. (E) Pooled cells from one month BCG vaccinated IL-21R knockout and respective control mice were cultured with or without Ag85. In all panels, after five days, expansion of CD3-NKp46+CD27+ cells were measured by flow cytometry. Mean values and SEs are shown. Data are representative oftwo independent experiments.

    Journal: Mucosal immunology

    Article Title: IL-21-dependent expansion of memory-like NK cells enhances protective immune responses against Mycobacterium tuberculosis

    doi: 10.1038/mi.2016.105

    Figure Lengend Snippet: Expansion of memory-like NK cells in BCG-vaccinated mice depends on IL-21 C57BL/6 mice were treated with PBS or immunized subcutaneously with 10 6 CFU of BCG in 100 µl of PBS. One month after vaccination, spleen, and peripheral lymph node cells were pooled. (A) CD3- NK cells were isolated and cultured with peritoneal macrophages, with or without Ag85. (B) Pooled cells were cultured with or without Ag85, in the presence of isotype-matched control antibodies or antibodies to IL-4, IL-7, IL-17, or IL-21. (C) Pooled cells from one month BCG vaccinated C57BL/6 mice were transfected with either IL-21 or scrambled siRNA (control siRNA) and cultured with or without Ag85. (D) Pooled cells from one month BCG vaccinated IL-21 knockout and respective control mice were cultured with or without Ag85. (E) Pooled cells from one month BCG vaccinated IL-21R knockout and respective control mice were cultured with or without Ag85. In all panels, after five days, expansion of CD3-NKp46+CD27+ cells were measured by flow cytometry. Mean values and SEs are shown. Data are representative oftwo independent experiments.

    Article Snippet: Abs and other reagents For flow cytometry, we used FITC anti-CD3, PE anti-CD27, PE/Cy7 anti-NKp46, FITC anti-CD8, PE anti-CD11b, PE anti-CD56, allophycocyanin KLRG1, (all from BioLegend).

    Techniques: Mouse Assay, Isolation, Cell Culture, Transfection, Knock-Out, Flow Cytometry, Cytometry

    Expansion of memory-like NK cells in individuals with LTBI PBMC from 5 individuals with LTBI and 5 individuals without LTBI were labeled with CFSE and cultured, with or without γ- M. tb . After 5 days, proliferating CD3-CD56+CD27+ cells were measured by flow cytometry. (A) A representative flow cytometry plot is shown. NK cells were identified by sequentially gating on lymphocytic singlet population and then on CD3−CD56+ NK cells. The events within the gated CD3-CD56+ NK cells were analyzed for CFSE+ cells and plotted in the histograms. Total PBMC CFSE+CD3-CD56+CD27+ NK cell numbers are shown. (B) Absolute number of proliferating CD3-CD56+CD27+ cells. (C) Absolute number of CD3-CD56+CD27+ IFN-γ cells. Five independent experiments each time with 1 LTBI+ and one LTBI- donor was performed in panel A, B and C. (D) PBMC from 5 individuals with LTBI were cultured, with or without γ- M. tb . After 3 days, CD3-CD56+CD27+ and CD3-CD56+CD27- cells were isolated by magnetic selection. CD14 + monocytes (10 6 /well) were isolated from fresh PBMC and differentiate them to macrophages (MDMs) for 3 days. MDMs were infected with M. tb H37Rv at a MOI of 1:2.5 (2.5 M. tb to one MDM). To some wells, the above isolated CD3-CD56+CD27+ or CD3-CD56+CD27- cells were added, at a ratio of 1 NK cell:9 MDMs. Infected macrophages were cultured for 5 days, and bacterial burden was determined. Mean values and SEs are shown.The data shown in panel D was performed six times, each time with PBMC obtained from one LTBI+ donor.

    Journal: Mucosal immunology

    Article Title: IL-21-dependent expansion of memory-like NK cells enhances protective immune responses against Mycobacterium tuberculosis

    doi: 10.1038/mi.2016.105

    Figure Lengend Snippet: Expansion of memory-like NK cells in individuals with LTBI PBMC from 5 individuals with LTBI and 5 individuals without LTBI were labeled with CFSE and cultured, with or without γ- M. tb . After 5 days, proliferating CD3-CD56+CD27+ cells were measured by flow cytometry. (A) A representative flow cytometry plot is shown. NK cells were identified by sequentially gating on lymphocytic singlet population and then on CD3−CD56+ NK cells. The events within the gated CD3-CD56+ NK cells were analyzed for CFSE+ cells and plotted in the histograms. Total PBMC CFSE+CD3-CD56+CD27+ NK cell numbers are shown. (B) Absolute number of proliferating CD3-CD56+CD27+ cells. (C) Absolute number of CD3-CD56+CD27+ IFN-γ cells. Five independent experiments each time with 1 LTBI+ and one LTBI- donor was performed in panel A, B and C. (D) PBMC from 5 individuals with LTBI were cultured, with or without γ- M. tb . After 3 days, CD3-CD56+CD27+ and CD3-CD56+CD27- cells were isolated by magnetic selection. CD14 + monocytes (10 6 /well) were isolated from fresh PBMC and differentiate them to macrophages (MDMs) for 3 days. MDMs were infected with M. tb H37Rv at a MOI of 1:2.5 (2.5 M. tb to one MDM). To some wells, the above isolated CD3-CD56+CD27+ or CD3-CD56+CD27- cells were added, at a ratio of 1 NK cell:9 MDMs. Infected macrophages were cultured for 5 days, and bacterial burden was determined. Mean values and SEs are shown.The data shown in panel D was performed six times, each time with PBMC obtained from one LTBI+ donor.

    Article Snippet: Abs and other reagents For flow cytometry, we used FITC anti-CD3, PE anti-CD27, PE/Cy7 anti-NKp46, FITC anti-CD8, PE anti-CD11b, PE anti-CD56, allophycocyanin KLRG1, (all from BioLegend).

    Techniques: Labeling, Cell Culture, Flow Cytometry, Cytometry, Isolation, Selection, Infection

    Memory-like NK cells expand after BCG vaccination and challenge with M. tb H37Rv C57BL/6 mice (20 mice per group) were given 100 µl of PBS or immunized subcutaneously with 10 6 CFU of BCG in 100 µl of PBS. After thirty days, mice were challenged with 75–100 CFU of M. tb H37Rv by aerosol. At weekly intervals up to 4 weeks, five mice in each group were sacrificed, and the lung bacterial burden and percentages of CD3-NKp46+ cells in lungs and spleen that were CD27+ were determined. (A) CD3-NKp46+CD27+ cells in lungs. (B) CD3-NKp46+CD27+ cells in spleens. (C) A representative flow cytometry plot is shown. Gating strategy to identify NK cells was similar to Figure 1 . (D) Bacterial burden in lungs. Mean values and SEs are shown. Data are representative of two independent experiments.

    Journal: Mucosal immunology

    Article Title: IL-21-dependent expansion of memory-like NK cells enhances protective immune responses against Mycobacterium tuberculosis

    doi: 10.1038/mi.2016.105

    Figure Lengend Snippet: Memory-like NK cells expand after BCG vaccination and challenge with M. tb H37Rv C57BL/6 mice (20 mice per group) were given 100 µl of PBS or immunized subcutaneously with 10 6 CFU of BCG in 100 µl of PBS. After thirty days, mice were challenged with 75–100 CFU of M. tb H37Rv by aerosol. At weekly intervals up to 4 weeks, five mice in each group were sacrificed, and the lung bacterial burden and percentages of CD3-NKp46+ cells in lungs and spleen that were CD27+ were determined. (A) CD3-NKp46+CD27+ cells in lungs. (B) CD3-NKp46+CD27+ cells in spleens. (C) A representative flow cytometry plot is shown. Gating strategy to identify NK cells was similar to Figure 1 . (D) Bacterial burden in lungs. Mean values and SEs are shown. Data are representative of two independent experiments.

    Article Snippet: Abs and other reagents For flow cytometry, we used FITC anti-CD3, PE anti-CD27, PE/Cy7 anti-NKp46, FITC anti-CD8, PE anti-CD11b, PE anti-CD56, allophycocyanin KLRG1, (all from BioLegend).

    Techniques: Mouse Assay, Flow Cytometry, Cytometry

    Memory-like NK cells proliferate and produce IFN-γ in M. tb infected mice C57BL/6 (CD45.2+ congenic) mice were given 100µl PBS or immunized subcutaneously with 10 6 CFU of BCG in 100µl PBS. Six months after vaccination, CD3-NKp46+CD27+ NK cells were isolated from pooled spleens and peripheral lymph node cells. 1 × 10 6 cells were adoptively transferred to CD45.1 mice (5 mice per group) through tail vein injection 10 days prior to infection with M. tb H37Rv. (A) Schematic representation of the adoptive transfer experiment ( B ) A representative flow cytometry plot is shown. Gating strategy to identify NK cells was similar to Figure 1 . (C) Absolute number of adoptively transferred CD45.2+ NK cells in lungs were determined at day 0, 15 and 30 after infection (D) Absolute number of adoptively transferred CD45.2+NKp46+CD27+ NK cells in lungs were determined at day 0, 15 and 30 after infection and (E) Absolute number of adoptively transferred CD45.2+NKp46+CD27+IFN-γ+ NK cells in lungs were determined at day 0, 15 and 30 after infection. Mean values and SEs are shown. Data are representative of two independent experiments.

    Journal: Mucosal immunology

    Article Title: IL-21-dependent expansion of memory-like NK cells enhances protective immune responses against Mycobacterium tuberculosis

    doi: 10.1038/mi.2016.105

    Figure Lengend Snippet: Memory-like NK cells proliferate and produce IFN-γ in M. tb infected mice C57BL/6 (CD45.2+ congenic) mice were given 100µl PBS or immunized subcutaneously with 10 6 CFU of BCG in 100µl PBS. Six months after vaccination, CD3-NKp46+CD27+ NK cells were isolated from pooled spleens and peripheral lymph node cells. 1 × 10 6 cells were adoptively transferred to CD45.1 mice (5 mice per group) through tail vein injection 10 days prior to infection with M. tb H37Rv. (A) Schematic representation of the adoptive transfer experiment ( B ) A representative flow cytometry plot is shown. Gating strategy to identify NK cells was similar to Figure 1 . (C) Absolute number of adoptively transferred CD45.2+ NK cells in lungs were determined at day 0, 15 and 30 after infection (D) Absolute number of adoptively transferred CD45.2+NKp46+CD27+ NK cells in lungs were determined at day 0, 15 and 30 after infection and (E) Absolute number of adoptively transferred CD45.2+NKp46+CD27+IFN-γ+ NK cells in lungs were determined at day 0, 15 and 30 after infection. Mean values and SEs are shown. Data are representative of two independent experiments.

    Article Snippet: Abs and other reagents For flow cytometry, we used FITC anti-CD3, PE anti-CD27, PE/Cy7 anti-NKp46, FITC anti-CD8, PE anti-CD11b, PE anti-CD56, allophycocyanin KLRG1, (all from BioLegend).

    Techniques: Infection, Mouse Assay, Isolation, Injection, Adoptive Transfer Assay, Flow Cytometry, Cytometry

    CD3-NKp46+CD27+ and CD3-NKp46+CD27+KLRG1+ NK cells protects mice from M. tb infection (A) Wild type C57BL/6 mice (5 mice per group) were immunized subcutaneously with 10 6 CFU of BCG in 100 µl of PBS or treated with PBS. After one month, CD3-NKp46+CD27+ or CD3-NKp46+CD27- NK cells from pooled spleens and peripheral lymph node cells were isolated and adoptively transferred (1 × 10 6 cells once on day 0 of infection) to M. tb H37Rv-infected C57BL/6 mice. (B) The same experiment was performed as in panel A, except that M. tb -infected wild type mice received CD3-NKp46+CD27+KLRG1+ or CD3-NKp46+CD27+KLRG1- NK cells from BCG vaccinated C57BL/6 mice. Infected mice in both panels were sacrificed thirty days post-infection, lung bacterial burden was measured. Mean values and SEs are shown. Data are representative of two independent experiments.

    Journal: Mucosal immunology

    Article Title: IL-21-dependent expansion of memory-like NK cells enhances protective immune responses against Mycobacterium tuberculosis

    doi: 10.1038/mi.2016.105

    Figure Lengend Snippet: CD3-NKp46+CD27+ and CD3-NKp46+CD27+KLRG1+ NK cells protects mice from M. tb infection (A) Wild type C57BL/6 mice (5 mice per group) were immunized subcutaneously with 10 6 CFU of BCG in 100 µl of PBS or treated with PBS. After one month, CD3-NKp46+CD27+ or CD3-NKp46+CD27- NK cells from pooled spleens and peripheral lymph node cells were isolated and adoptively transferred (1 × 10 6 cells once on day 0 of infection) to M. tb H37Rv-infected C57BL/6 mice. (B) The same experiment was performed as in panel A, except that M. tb -infected wild type mice received CD3-NKp46+CD27+KLRG1+ or CD3-NKp46+CD27+KLRG1- NK cells from BCG vaccinated C57BL/6 mice. Infected mice in both panels were sacrificed thirty days post-infection, lung bacterial burden was measured. Mean values and SEs are shown. Data are representative of two independent experiments.

    Article Snippet: Abs and other reagents For flow cytometry, we used FITC anti-CD3, PE anti-CD27, PE/Cy7 anti-NKp46, FITC anti-CD8, PE anti-CD11b, PE anti-CD56, allophycocyanin KLRG1, (all from BioLegend).

    Techniques: Mouse Assay, Infection, Isolation

    IL-21 is required at the time of BCG vaccination for the generation of memory-like NK cells C57BL/6 (Rag2 knockout) mice were treated with PBS or immunized subcutaneously with 10 6 CFU of BCG in 100 µl of PBS. Some of the BCG vaccinated mice received 0.3 mg of recombinant IL-21 or PBS through tail vein at the time of BCG vaccination. One month after vaccination, spleen, and peripheral lymph node cells were pooled. (A) Spleen, and peripheral lymph node cells from the above groups of mice were cultured in the presence or absence of Ag85. After 5 days, expansion of CD3-NKp46+CD27+ cells was determined by flow cytometry. (B) Rag2 Knockout mice (5 mice per group) were immunized subcutaneously with 10 6 CFU of BCG and treated with or without recombinant IL-21. After one month, CD3-NKp46+CD27+ NK cells from pooled spleens and peripheral lymph node cells were isolated and adoptively transferred (1 × 10 6 cells once on day 0 of infection) to M. tb H37Rv-infected C57BL/6 mice. Infected mice were sacrificed thirty days post-infection, lung bacterial burden was measured. Mean values and SEs are shown. Data are representative of three independent experiments.

    Journal: Mucosal immunology

    Article Title: IL-21-dependent expansion of memory-like NK cells enhances protective immune responses against Mycobacterium tuberculosis

    doi: 10.1038/mi.2016.105

    Figure Lengend Snippet: IL-21 is required at the time of BCG vaccination for the generation of memory-like NK cells C57BL/6 (Rag2 knockout) mice were treated with PBS or immunized subcutaneously with 10 6 CFU of BCG in 100 µl of PBS. Some of the BCG vaccinated mice received 0.3 mg of recombinant IL-21 or PBS through tail vein at the time of BCG vaccination. One month after vaccination, spleen, and peripheral lymph node cells were pooled. (A) Spleen, and peripheral lymph node cells from the above groups of mice were cultured in the presence or absence of Ag85. After 5 days, expansion of CD3-NKp46+CD27+ cells was determined by flow cytometry. (B) Rag2 Knockout mice (5 mice per group) were immunized subcutaneously with 10 6 CFU of BCG and treated with or without recombinant IL-21. After one month, CD3-NKp46+CD27+ NK cells from pooled spleens and peripheral lymph node cells were isolated and adoptively transferred (1 × 10 6 cells once on day 0 of infection) to M. tb H37Rv-infected C57BL/6 mice. Infected mice were sacrificed thirty days post-infection, lung bacterial burden was measured. Mean values and SEs are shown. Data are representative of three independent experiments.

    Article Snippet: Abs and other reagents For flow cytometry, we used FITC anti-CD3, PE anti-CD27, PE/Cy7 anti-NKp46, FITC anti-CD8, PE anti-CD11b, PE anti-CD56, allophycocyanin KLRG1, (all from BioLegend).

    Techniques: Knock-Out, Mouse Assay, Recombinant, Cell Culture, Flow Cytometry, Cytometry, Isolation, Infection

    BCG vaccination induces expansion of memory-like NK cells ( A, B ) C57BL/6 mice (5 mice per group) were given 100 µl of PBS (unimmunized) or immunized subcutaneously with 10 6 CFU of BCG in 100 µl of PBS. Six months after vaccination, spleen, and peripheral lymph node cells were isolated, pooled, labeled with carboxyfluorescein succinimidyl ester (CFSE) and cultured, with or without γ- M. tb or heat killed Candida albicans . After 5 days, expanding CD3-NKp46+CD27+ NK cells and IFN-γ producing cells were measured by flow cytometry. (A) A representative flow cytometry plot is shown. NK cells were identified by sequentially gating on singlet population and then on CD3− NKp46+ NK cells. The events within the gated CD3-NKp46+ NK cells were analyzed for CFSE+ cells and plotted in the histograms. Total lung CFSE+CD3-NKp46+CD27+ NK cell numbers are shown. (B) Percent proliferating NK cells (C) Absolute number of CD3-NKp46+CD27+ cells (D) CD3-NKp46+CD27-IFN-γ+ and CD3-NKp46+CD27+IFN-γ+ cells. Mean values and SEs are shown. Data are representative of two independent experiments.

    Journal: Mucosal immunology

    Article Title: IL-21-dependent expansion of memory-like NK cells enhances protective immune responses against Mycobacterium tuberculosis

    doi: 10.1038/mi.2016.105

    Figure Lengend Snippet: BCG vaccination induces expansion of memory-like NK cells ( A, B ) C57BL/6 mice (5 mice per group) were given 100 µl of PBS (unimmunized) or immunized subcutaneously with 10 6 CFU of BCG in 100 µl of PBS. Six months after vaccination, spleen, and peripheral lymph node cells were isolated, pooled, labeled with carboxyfluorescein succinimidyl ester (CFSE) and cultured, with or without γ- M. tb or heat killed Candida albicans . After 5 days, expanding CD3-NKp46+CD27+ NK cells and IFN-γ producing cells were measured by flow cytometry. (A) A representative flow cytometry plot is shown. NK cells were identified by sequentially gating on singlet population and then on CD3− NKp46+ NK cells. The events within the gated CD3-NKp46+ NK cells were analyzed for CFSE+ cells and plotted in the histograms. Total lung CFSE+CD3-NKp46+CD27+ NK cell numbers are shown. (B) Percent proliferating NK cells (C) Absolute number of CD3-NKp46+CD27+ cells (D) CD3-NKp46+CD27-IFN-γ+ and CD3-NKp46+CD27+IFN-γ+ cells. Mean values and SEs are shown. Data are representative of two independent experiments.

    Article Snippet: Abs and other reagents For flow cytometry, we used FITC anti-CD3, PE anti-CD27, PE/Cy7 anti-NKp46, FITC anti-CD8, PE anti-CD11b, PE anti-CD56, allophycocyanin KLRG1, (all from BioLegend).

    Techniques: Mouse Assay, Isolation, Labeling, Cell Culture, Flow Cytometry, Cytometry

    Memory B cells and PSGL-1 hi PD-1 hi CXCR5 hi cells are enriched at the tonsillar T-B border (A) CD27 (red), CD19 (green), and CD38 (blue) confocal staining. Arrows in merged picture indicate CD19 + CD27 hi CD38 − (yellow) cells. Cells in the box are magnified on top upper right. Scale bar = 50 μm. (B) PSGL-1 (red), CD19 (green), and PD-1 (blue) confocal staining. Arrows in merged picture indicates CD19 − PSGL-1 hi PD-1 hi (purple) cells. Cells in the box are magnified on top upper right. Scale bar = 50 μm. (C) Numbers of CD19 + CD27 hi CD38 − cells (left panel) and CD19 − PSGL-1 hi PD-1 hi (middle panel) cells per mm 2 in indicated anatomic regions. Two graphs are merged (right panel). GC: germinal center, F: follicular mantle, T-B border: Border of the T cell zone and B cell follicle, TZ: T cell zone, SE: subepithelial area. Left panel: N = 13 images from 3 different tonsils, except the subepithelial area, in which N = 9. Middle panel; N = 11 images from 3 different tonsils, except subepithelial area, in which N = 6. One-way ANOVA test. *** P

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

    Article Title: Human Extrafollicular CD4+ T Helper Cells Help Memory B Cells Produce Immunoglobulins

    doi: 10.4049/jimmunol.1701217

    Figure Lengend Snippet: Memory B cells and PSGL-1 hi PD-1 hi CXCR5 hi cells are enriched at the tonsillar T-B border (A) CD27 (red), CD19 (green), and CD38 (blue) confocal staining. Arrows in merged picture indicate CD19 + CD27 hi CD38 − (yellow) cells. Cells in the box are magnified on top upper right. Scale bar = 50 μm. (B) PSGL-1 (red), CD19 (green), and PD-1 (blue) confocal staining. Arrows in merged picture indicates CD19 − PSGL-1 hi PD-1 hi (purple) cells. Cells in the box are magnified on top upper right. Scale bar = 50 μm. (C) Numbers of CD19 + CD27 hi CD38 − cells (left panel) and CD19 − PSGL-1 hi PD-1 hi (middle panel) cells per mm 2 in indicated anatomic regions. Two graphs are merged (right panel). GC: germinal center, F: follicular mantle, T-B border: Border of the T cell zone and B cell follicle, TZ: T cell zone, SE: subepithelial area. Left panel: N = 13 images from 3 different tonsils, except the subepithelial area, in which N = 9. Middle panel; N = 11 images from 3 different tonsils, except subepithelial area, in which N = 6. One-way ANOVA test. *** P

    Article Snippet: After blocking with rat serum for 20 minutes, slides were stained with anti-CD19 FITC (HIB19, BD, 1:25 dilution), anti-IgD FITC (IA6-2, BD, 1:25 dilution) or PE (IgD26, Miltenyi Biotec, 1:25 dilution), anti-CD27 PE (O323, Biolegend, 1:25 dilution) or Alexa Fluor 647 (O323, Biolegend, 1:25 dilution), anti-CD38 Alexa Fluor 647 (HIT2, Biolegend, 1:10 dilution), anti-PSGL-1 PE (KPL-1, BD, 1:10 dilution), and anti-CD4 AL647 (OKT4, Biolegend, 1:25 dilution).

    Techniques: Staining

    CD81 engagement induces the preferential expansion of naïve B lymphocytes. Purified naive (CD27 - ) or memory (CD27 + ) B cells were incubated with complete medium (Control), MG81 and N81 mAbs (5 μg/ml each) (CD81), or SAC (1:30,000, vol/vol).

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

    Article Title: Activation of na?ve B lymphocytes via CD81, a pathogenetic mechanism for hepatitis C virus-associated B lymphocyte disorders

    doi: 10.1073/pnas.0509402102

    Figure Lengend Snippet: CD81 engagement induces the preferential expansion of naïve B lymphocytes. Purified naive (CD27 - ) or memory (CD27 + ) B cells were incubated with complete medium (Control), MG81 and N81 mAbs (5 μg/ml each) (CD81), or SAC (1:30,000, vol/vol).

    Article Snippet: The CFSE-labeled cells were incubated with complete medium, anti-CD81 mAbs (MG81 and N81), or SAC for 96 h, stained with PE-anti-CD27, and analyzed by flow cytometry by using FACSCalibur (Becton Dickinson).

    Techniques: Purification, Incubation

    TB patients have increased percentages of CD27 low IFN-γ + CD4 T cells in their blood. A–C, Strategies for determining percentages of CD27 low (A), IFN-γ + (B) and CD27 low IFN-γ + (C) CD4 T cells. A, CD27 low cells were gated within the total population of CD4 + T cells. B, To identify IFN-γ + CD4 T cells, an aliquot of blood was stimulated with Mtb sonicate; another aliquote was left un-stimulated. During the analysis, the gates for IFN-γ + cells in Mtb -stimulated samples were plotted based on Mtb un-stimulated samples (Fig. B, dotted line). To identify CD27 low IFN-γ + cells, the expression of CD27 was first analyzed in IFN-γ − population. Because this population was always numerous, CD27 low and CD27 hi cells could be easily separated. The gates for CD27 low cells were then applied to IFN-γ + population (C, dotted line). D–F, Percentages of CD27 low (D), IFN-γ + (E), and CD27 low IFN-γ + (F) cells in TB patients (n = 50), TB contacts (n = 21) and Mtb -unexposed individuals (n = 15). G, Lack of correlation between the percentages of IFN-γ + and CD27 low IFN-γ + cells in TB patients, TB contacts and Mtb -unexposed individuals (n = 86). H, ROC-curve of CD27 low IFN- γ + cell percentages for discriminating TB patients from healthy individuals (TB contacts and Mtb -unexposed). I, Percentages of CD27 low IFN-γ + cells in TB contacts with positive and negative results of QFT assay *p

    Journal: PLoS ONE

    Article Title: Mtb-Specific CD27low CD4 T Cells as Markers of Lung Tissue Destruction during Pulmonary Tuberculosis in Humans

    doi: 10.1371/journal.pone.0043733

    Figure Lengend Snippet: TB patients have increased percentages of CD27 low IFN-γ + CD4 T cells in their blood. A–C, Strategies for determining percentages of CD27 low (A), IFN-γ + (B) and CD27 low IFN-γ + (C) CD4 T cells. A, CD27 low cells were gated within the total population of CD4 + T cells. B, To identify IFN-γ + CD4 T cells, an aliquot of blood was stimulated with Mtb sonicate; another aliquote was left un-stimulated. During the analysis, the gates for IFN-γ + cells in Mtb -stimulated samples were plotted based on Mtb un-stimulated samples (Fig. B, dotted line). To identify CD27 low IFN-γ + cells, the expression of CD27 was first analyzed in IFN-γ − population. Because this population was always numerous, CD27 low and CD27 hi cells could be easily separated. The gates for CD27 low cells were then applied to IFN-γ + population (C, dotted line). D–F, Percentages of CD27 low (D), IFN-γ + (E), and CD27 low IFN-γ + (F) cells in TB patients (n = 50), TB contacts (n = 21) and Mtb -unexposed individuals (n = 15). G, Lack of correlation between the percentages of IFN-γ + and CD27 low IFN-γ + cells in TB patients, TB contacts and Mtb -unexposed individuals (n = 86). H, ROC-curve of CD27 low IFN- γ + cell percentages for discriminating TB patients from healthy individuals (TB contacts and Mtb -unexposed). I, Percentages of CD27 low IFN-γ + cells in TB contacts with positive and negative results of QFT assay *p

    Article Snippet: For CD27low cells, 200 ul of freshly isolated blood were stained with PerCP-Cy5.5 anti-CD4 and PE-anti-CD27 mAbs (BD Biosciences, San Jose, USA; e-bioscience, San Diego, USA; 10 min, room temperature).

    Techniques: Expressing

    Association between blood CD27 low IFN-γ + cells and different manifestations of TB disease. A–F, Percentages of CD27 low IFN-γ + cells in TB patients (n = 50) grouped based on different characteristics of TB disease. For multiple (seven) parameter testing, p- value

    Journal: PLoS ONE

    Article Title: Mtb-Specific CD27low CD4 T Cells as Markers of Lung Tissue Destruction during Pulmonary Tuberculosis in Humans

    doi: 10.1371/journal.pone.0043733

    Figure Lengend Snippet: Association between blood CD27 low IFN-γ + cells and different manifestations of TB disease. A–F, Percentages of CD27 low IFN-γ + cells in TB patients (n = 50) grouped based on different characteristics of TB disease. For multiple (seven) parameter testing, p- value

    Article Snippet: For CD27low cells, 200 ul of freshly isolated blood were stained with PerCP-Cy5.5 anti-CD4 and PE-anti-CD27 mAbs (BD Biosciences, San Jose, USA; e-bioscience, San Diego, USA; 10 min, room temperature).

    Techniques:

    Following anti-TB therapy, CD27 low IFN-γ + cells decline parallel to reduction/repair of lung destruction. A, Percentages of CD27 low IFN-γ + cells were determined at the start of treatment and two months later. Depending on the results of “IFN-γ/CD27” assay, patients were divided into three groups: in “CD27 low - high” group, initially elevated percentages of CD27 low IFN-γ + cells did not decline following 2-mo therapy; in “CD27 low – reduced” group, percentages of CD27 low IFN-γ + cells declined to become below the 47% threshold, but remained above 35.1% threshold (upper limit of norm); in “CD27 low - normalized” group, percentages of CD27 low IFN-γ + cells declined to become below the 35.1% threshold. Dotted lines show 35.1 and 47% thresholds. B–D, diagrams showing changes in lung destruction (B), sputum Mtb -positivity (C) and clinical TB severity (D) for each group of patients following 2-mo therapy. Closed segments, no improvement; squared segments, reduction of lung destruction, clinical symptoms or numbers of Mtb in the sputum; open segments, normalization (repair of lung destruction, conversion of sputum assay, disappearance of intoxication symptoms), striped segments – no abnormalities at the start of the treatment.

    Journal: PLoS ONE

    Article Title: Mtb-Specific CD27low CD4 T Cells as Markers of Lung Tissue Destruction during Pulmonary Tuberculosis in Humans

    doi: 10.1371/journal.pone.0043733

    Figure Lengend Snippet: Following anti-TB therapy, CD27 low IFN-γ + cells decline parallel to reduction/repair of lung destruction. A, Percentages of CD27 low IFN-γ + cells were determined at the start of treatment and two months later. Depending on the results of “IFN-γ/CD27” assay, patients were divided into three groups: in “CD27 low - high” group, initially elevated percentages of CD27 low IFN-γ + cells did not decline following 2-mo therapy; in “CD27 low – reduced” group, percentages of CD27 low IFN-γ + cells declined to become below the 47% threshold, but remained above 35.1% threshold (upper limit of norm); in “CD27 low - normalized” group, percentages of CD27 low IFN-γ + cells declined to become below the 35.1% threshold. Dotted lines show 35.1 and 47% thresholds. B–D, diagrams showing changes in lung destruction (B), sputum Mtb -positivity (C) and clinical TB severity (D) for each group of patients following 2-mo therapy. Closed segments, no improvement; squared segments, reduction of lung destruction, clinical symptoms or numbers of Mtb in the sputum; open segments, normalization (repair of lung destruction, conversion of sputum assay, disappearance of intoxication symptoms), striped segments – no abnormalities at the start of the treatment.

    Article Snippet: For CD27low cells, 200 ul of freshly isolated blood were stained with PerCP-Cy5.5 anti-CD4 and PE-anti-CD27 mAbs (BD Biosciences, San Jose, USA; e-bioscience, San Diego, USA; 10 min, room temperature).

    Techniques:

    B cell subsets. ( a ) The percentage of B cells (CD3− CD19+) was determined after staining of PBMC, derived on days 0 and 35, with CD3, CD19, CD27 and IgD and gating on the live lymphocyte population in a SSC/FSC blot. ( b ) Further evaluation of naive (CD27− IgD+), ( c ) unswitched (CD27+IgD+) ( d ) and switched memory B (CD27+IgD−) cells were performed according to the expression of CD27 and IgD on gated B cells. Statistical analysis by General Linear Model with arcsine-transformed percentages. Individual comparisons by linear contrasts. * p

    Journal: Scientific Reports

    Article Title: Age-related differences in humoral and cellular immune responses after primary immunisation: indications for stratified vaccination schedules

    doi: 10.1038/s41598-018-28111-8

    Figure Lengend Snippet: B cell subsets. ( a ) The percentage of B cells (CD3− CD19+) was determined after staining of PBMC, derived on days 0 and 35, with CD3, CD19, CD27 and IgD and gating on the live lymphocyte population in a SSC/FSC blot. ( b ) Further evaluation of naive (CD27− IgD+), ( c ) unswitched (CD27+IgD+) ( d ) and switched memory B (CD27+IgD−) cells were performed according to the expression of CD27 and IgD on gated B cells. Statistical analysis by General Linear Model with arcsine-transformed percentages. Individual comparisons by linear contrasts. * p

    Article Snippet: In order to distinguish between different B-cell and T-cell subsets, the following monoclonal antibodies were added for surface staining: anti-CD3-Pe-Cy5 (BD Pharmingen), anti-CD4 FITC (BD), anti-CD4-PE-Cy5 (BD), anti-CD8-APC (BD), anti-CD19-FITC (BD), anti-CD24-PE (BioLegend, San Diego, CA, USA), anti-CD27 PE (BD), anti-CD28-PE-Cy5 (BD), anti-CD38-PE-Cy5 (BioLegend), anti-CD45RA-PE (BD) and anti-CCR7-FITC (R & D Systems).

    Techniques: Staining, Derivative Assay, Expressing, Transformation Assay

    The glutamine-α-KG axis is involved in the dysfunction in menin KO CD8 T cells. a A representative staining profile of CD62L/CD27 on the cell surface of the WT and menin KO effector CD8 T cells on day 7. The percentages of cells are indicated in each quadrant. Naive CD8 T cells were activated and cultured for 3 days under normal (Ctrl), glutamine-deprived (dGln), or glutamine-deprived supplemented with DM-α-KG (dGln/α-KG) conditions for 3 days, and then the cells were further expanded with IL-2 under normal conditions for an additional 4 days. An analysis was performed on day 7 after the initial anti-TCR-β/CD28 stimulation. b Representative staining profiles of CD226 and PD-1 on the cell surface of the cells in a . c The ELISA results for IL-6, IL-10, and OPN in the supernatants of the cells in a restimulated with immobilized anti-TCR-β for 16 h are shown with standard deviation ( n = 3: biological replicates). d The results of the quantitative RT-PCR analysis of the pro-inflammatory enzymes in the cells in a . The results are presented relative to the expression of Cd3ε mRNA with the standard deviations ( n = 3: technical replicates). e The percentages of SA β-galactosidase (SA β-Gal)-positive cells on day 12 after the initial anti-TCR-β/CD28 mAb stimulation are shown with the standard deviation ( n = 3: biological replicates). f A 1:1 mixture of WT OT-1 Tg effector CD8 T (Thy1.1 + )/ menin KO OT-1 Tg effector CD8 T cells under normal conditions (Thy1.2 + ) or WT (Thy1.1 + )/ menin KO under glutamine-deprived conditions (Thy1.2 + ) was adoptively transferred into WT congenic (Thy1.1 + Thy1.2 + ) mice. Twenty days after the transfer, the mice were infected with Lm -OVA to activate the donor cells. The donor cells were collected from the spleen on day 5 after Lm -OVA infection and analyzed by FACS. The absolute number of donor cells in the spleen is shown (mean ± SD, n = 4 per group: biological replicates). * p

    Journal: Nature Communications

    Article Title: The tumor suppressor menin prevents effector CD8 T-cell dysfunction by targeting mTORC1-dependent metabolic activation

    doi: 10.1038/s41467-018-05854-6

    Figure Lengend Snippet: The glutamine-α-KG axis is involved in the dysfunction in menin KO CD8 T cells. a A representative staining profile of CD62L/CD27 on the cell surface of the WT and menin KO effector CD8 T cells on day 7. The percentages of cells are indicated in each quadrant. Naive CD8 T cells were activated and cultured for 3 days under normal (Ctrl), glutamine-deprived (dGln), or glutamine-deprived supplemented with DM-α-KG (dGln/α-KG) conditions for 3 days, and then the cells were further expanded with IL-2 under normal conditions for an additional 4 days. An analysis was performed on day 7 after the initial anti-TCR-β/CD28 stimulation. b Representative staining profiles of CD226 and PD-1 on the cell surface of the cells in a . c The ELISA results for IL-6, IL-10, and OPN in the supernatants of the cells in a restimulated with immobilized anti-TCR-β for 16 h are shown with standard deviation ( n = 3: biological replicates). d The results of the quantitative RT-PCR analysis of the pro-inflammatory enzymes in the cells in a . The results are presented relative to the expression of Cd3ε mRNA with the standard deviations ( n = 3: technical replicates). e The percentages of SA β-galactosidase (SA β-Gal)-positive cells on day 12 after the initial anti-TCR-β/CD28 mAb stimulation are shown with the standard deviation ( n = 3: biological replicates). f A 1:1 mixture of WT OT-1 Tg effector CD8 T (Thy1.1 + )/ menin KO OT-1 Tg effector CD8 T cells under normal conditions (Thy1.2 + ) or WT (Thy1.1 + )/ menin KO under glutamine-deprived conditions (Thy1.2 + ) was adoptively transferred into WT congenic (Thy1.1 + Thy1.2 + ) mice. Twenty days after the transfer, the mice were infected with Lm -OVA to activate the donor cells. The donor cells were collected from the spleen on day 5 after Lm -OVA infection and analyzed by FACS. The absolute number of donor cells in the spleen is shown (mean ± SD, n = 4 per group: biological replicates). * p

    Article Snippet: Antibodies used for intracellular and cell-surface staining were as follows: anti-Osteopontin-phycoerythrin (PE) mAb (cat#IC808P; R & D Systems, Minneapolis, MN, USA), anti-IFN-γ-allophycocyanin (APC) mAb (cat#554413; BD Bioscience, San Jose, CA, USA), anti-IFN-γ-fluorescein isothiocyanate (FITC) mAb (cat#554411; BD Bioscience), IL-2-APC mAb (cat#JES6-5H4; TONBO Biosciences, San Diego, CA, USA), anti-Phospho-S6 (Ser235/236)-Alexa Fluor 647 mAb (cat#4851; Cell Signaling Technology, Danvers, MA, USA), anti-Phospho-S6 (Ser240/244) Alexa Fluor 647 mAb (cat#5044; Cell Signaling Technology), anti-S6 ribosomal protein Alexa Fluor 647 mAb (cat#5548; Cell Signaling Technology), anti-CD27-PE mAb (cat#558754; BD Bioscience), anti-CD62L-APC mAb (cat#20-0621; TONBO Biosciences), anti-CD226-APC mAb (cat#128809; BioLegend, San Diego, CA, USA), and anti-PD-1-PE mAb (cat#551892; BD Bioscience).

    Techniques: Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation, Quantitative RT-PCR, Expressing, Mouse Assay, Infection, FACS

    Demethylation of histone H3K27 is involved in CD8 T-cell dysfunction. a The result of the immunoblot analysis of the di- or tri-methylated histone H3K27, tri-methylated histone H3K4 and total histone H3 in the WT CD8 T cells cultured under the indicated conditions for 48 h. The protein amount of histone H3 was used as a loading control. b The results of the immunoblot analysis of the di- or tri-methylated histone H3K27, tri-methylated histone H3K4 and total histone H3 in the WT or menin KO CD8 T cells cultured under normal conditions for 3 days. c A representative staining profile of CD62L/CD27 on the cell surface of the WT, utx KO menin KO or menin / utx -double KO effector CD8 T cells on day 7. The percentages of cells are indicated in each quadrant. d Representative staining profile of PD-1 on the cell surface of the cells in c . e The ELISA results for IL-6, IL-10, OPN, and IFN-γ in the supernatants of the cells in c restimulated with immobilized anti-TCR-β for 16 h are shown with the standard deviation ( n = 3: biological replicates). f The results of the quantitative RT-PCR analysis of the pro-inflammatory enzymes of the cells in c . The results are presented relative to the expression of Cd3ε mRNA with the standard deviations ( n = 3: technical replicates). g The percentages of SA β-galactosidase (SA β-Gal)-positive cells on day 12 after the initial anti-TCR-β/CD28 stimulation are shown with the standard deviation ( n = 3: biological replicates). * p

    Journal: Nature Communications

    Article Title: The tumor suppressor menin prevents effector CD8 T-cell dysfunction by targeting mTORC1-dependent metabolic activation

    doi: 10.1038/s41467-018-05854-6

    Figure Lengend Snippet: Demethylation of histone H3K27 is involved in CD8 T-cell dysfunction. a The result of the immunoblot analysis of the di- or tri-methylated histone H3K27, tri-methylated histone H3K4 and total histone H3 in the WT CD8 T cells cultured under the indicated conditions for 48 h. The protein amount of histone H3 was used as a loading control. b The results of the immunoblot analysis of the di- or tri-methylated histone H3K27, tri-methylated histone H3K4 and total histone H3 in the WT or menin KO CD8 T cells cultured under normal conditions for 3 days. c A representative staining profile of CD62L/CD27 on the cell surface of the WT, utx KO menin KO or menin / utx -double KO effector CD8 T cells on day 7. The percentages of cells are indicated in each quadrant. d Representative staining profile of PD-1 on the cell surface of the cells in c . e The ELISA results for IL-6, IL-10, OPN, and IFN-γ in the supernatants of the cells in c restimulated with immobilized anti-TCR-β for 16 h are shown with the standard deviation ( n = 3: biological replicates). f The results of the quantitative RT-PCR analysis of the pro-inflammatory enzymes of the cells in c . The results are presented relative to the expression of Cd3ε mRNA with the standard deviations ( n = 3: technical replicates). g The percentages of SA β-galactosidase (SA β-Gal)-positive cells on day 12 after the initial anti-TCR-β/CD28 stimulation are shown with the standard deviation ( n = 3: biological replicates). * p

    Article Snippet: Antibodies used for intracellular and cell-surface staining were as follows: anti-Osteopontin-phycoerythrin (PE) mAb (cat#IC808P; R & D Systems, Minneapolis, MN, USA), anti-IFN-γ-allophycocyanin (APC) mAb (cat#554413; BD Bioscience, San Jose, CA, USA), anti-IFN-γ-fluorescein isothiocyanate (FITC) mAb (cat#554411; BD Bioscience), IL-2-APC mAb (cat#JES6-5H4; TONBO Biosciences, San Diego, CA, USA), anti-Phospho-S6 (Ser235/236)-Alexa Fluor 647 mAb (cat#4851; Cell Signaling Technology, Danvers, MA, USA), anti-Phospho-S6 (Ser240/244) Alexa Fluor 647 mAb (cat#5044; Cell Signaling Technology), anti-S6 ribosomal protein Alexa Fluor 647 mAb (cat#5548; Cell Signaling Technology), anti-CD27-PE mAb (cat#558754; BD Bioscience), anti-CD62L-APC mAb (cat#20-0621; TONBO Biosciences), anti-CD226-APC mAb (cat#128809; BioLegend, San Diego, CA, USA), and anti-PD-1-PE mAb (cat#551892; BD Bioscience).

    Techniques: Methylation, Cell Culture, Staining, Enzyme-linked Immunosorbent Assay, Standard Deviation, Quantitative RT-PCR, Expressing

    Menin deficiency induces dysfunction of CD8 T cells. a A representative staining profile of CD62L/CD27 on the cell surface of the WT and menin KO effector CD8 T cells. Naive CD8 T cells were stimulated with anti-TCR-β mAb plus anti-CD28 mAb with IL-2 for 2 days, and then the cells were further expanded with IL-2 for an additional 5 days. An analysis was performed on day 7 after the initial stimulation. b A representative staining profile of PD-1 on the cell surface of the WT and menin KO CD8 T cells on day 7. c Representative results of the intracellular FACS analysis of IFN-γ/OPN in the WT and menin KO effector CD8 T cells on day 7. The percentages of cells are indicated in each quadrant. d The results of ELISA for IL-6, IL-10, and OPN in the supernatants of the cells in c restimulated with immobilized anti-TCR-β for 16 h are shown with the standard deviation ( n = 3: biological replicates). e The results of the quantitative RT-PCR analysis of mRNAs encoding pro-inflammatory enzymes in the WT and menin KO effector CD8 T cells on day 7. The results are presented relative to the mRNA expression of Cd3ε with the standard deviations ( n = 3: technical replicates). f The percentages of SA β-galactosidase (SA β-Gal)-positive cells on day 12 are shown with the standard deviation ( n = 3: biological replicates). g A representative staining profile of CD62L/CD27 on the cell surface of the WT and menin KO OT1 Tg splenic CD8 T cells on day 7 after Lm-OVA infection. h A representative staining profile of PD-1 on the cell surface of the cells in g . i Representative results of the intracellular FACS analysis of IFN-γ/OPN in the cells in g stimulated with an OVA-peptide (SIINFEKL) for 6 h. ** p

    Journal: Nature Communications

    Article Title: The tumor suppressor menin prevents effector CD8 T-cell dysfunction by targeting mTORC1-dependent metabolic activation

    doi: 10.1038/s41467-018-05854-6

    Figure Lengend Snippet: Menin deficiency induces dysfunction of CD8 T cells. a A representative staining profile of CD62L/CD27 on the cell surface of the WT and menin KO effector CD8 T cells. Naive CD8 T cells were stimulated with anti-TCR-β mAb plus anti-CD28 mAb with IL-2 for 2 days, and then the cells were further expanded with IL-2 for an additional 5 days. An analysis was performed on day 7 after the initial stimulation. b A representative staining profile of PD-1 on the cell surface of the WT and menin KO CD8 T cells on day 7. c Representative results of the intracellular FACS analysis of IFN-γ/OPN in the WT and menin KO effector CD8 T cells on day 7. The percentages of cells are indicated in each quadrant. d The results of ELISA for IL-6, IL-10, and OPN in the supernatants of the cells in c restimulated with immobilized anti-TCR-β for 16 h are shown with the standard deviation ( n = 3: biological replicates). e The results of the quantitative RT-PCR analysis of mRNAs encoding pro-inflammatory enzymes in the WT and menin KO effector CD8 T cells on day 7. The results are presented relative to the mRNA expression of Cd3ε with the standard deviations ( n = 3: technical replicates). f The percentages of SA β-galactosidase (SA β-Gal)-positive cells on day 12 are shown with the standard deviation ( n = 3: biological replicates). g A representative staining profile of CD62L/CD27 on the cell surface of the WT and menin KO OT1 Tg splenic CD8 T cells on day 7 after Lm-OVA infection. h A representative staining profile of PD-1 on the cell surface of the cells in g . i Representative results of the intracellular FACS analysis of IFN-γ/OPN in the cells in g stimulated with an OVA-peptide (SIINFEKL) for 6 h. ** p

    Article Snippet: Antibodies used for intracellular and cell-surface staining were as follows: anti-Osteopontin-phycoerythrin (PE) mAb (cat#IC808P; R & D Systems, Minneapolis, MN, USA), anti-IFN-γ-allophycocyanin (APC) mAb (cat#554413; BD Bioscience, San Jose, CA, USA), anti-IFN-γ-fluorescein isothiocyanate (FITC) mAb (cat#554411; BD Bioscience), IL-2-APC mAb (cat#JES6-5H4; TONBO Biosciences, San Diego, CA, USA), anti-Phospho-S6 (Ser235/236)-Alexa Fluor 647 mAb (cat#4851; Cell Signaling Technology, Danvers, MA, USA), anti-Phospho-S6 (Ser240/244) Alexa Fluor 647 mAb (cat#5044; Cell Signaling Technology), anti-S6 ribosomal protein Alexa Fluor 647 mAb (cat#5548; Cell Signaling Technology), anti-CD27-PE mAb (cat#558754; BD Bioscience), anti-CD62L-APC mAb (cat#20-0621; TONBO Biosciences), anti-CD226-APC mAb (cat#128809; BioLegend, San Diego, CA, USA), and anti-PD-1-PE mAb (cat#551892; BD Bioscience).

    Techniques: Staining, FACS, Enzyme-linked Immunosorbent Assay, Standard Deviation, Quantitative RT-PCR, Expressing, Infection

    Rapamycin inhibits dysfunction of menin KO CD8 T cells. a A representative staining profile of CD62L/CD27 on the cell surface of the WT and menin KO effector CD8 T cells on day 7. The percentages of cells are indicated in each quadrant. Naive CD8 T cells were stimulated with anti-TCR-β mAb plus anti-CD28 mAb with IL-2 in the presence or absence of rapamycin for 2 days, and then the cells were further expanded with IL-2 in the absence of rapamycin for an additional 5 days. b Representative staining profiles of CD226 and PD-1 on the surface of the cells in a . c The ELISA for IL-6, IL-10, and OPN in the supernatants of the cells in a restimulated with immobilized anti-TCR-β for 16 h are shown with the standard deviation ( n = 3: biological replicates). d The results of the quantitative RT-PCR analysis of the pro-inflammatory enzymes in the cells in a . The results are presented relative to the expression of Cd3ε mRNA with the standard deviations ( n = 3: technical replicates). e The percentages of SA β-galactosidase (SA β-Gal)-positive cells on day 12 are shown with the standard deviation ( n = 3: biological replicates). f A 1:1 mixture of WT OT-1 Tg effector CD8 T (Thy1.1 + )/ menin KO OT-1 Tg effector CD8 T cells (Thy1.2 + ) or WT (Thy1.1 + )/rapamycin-treated menin KO (Thy1.2 + ) was adoptively transferred into WT congenic (Thy1.1 + Thy1.2 + ) mice. Twenty days after the transfer, the mice were infected with Lm -OVA to activate the donor cells. The donor cells were collected from the spleen on day 5 after Lm -OVA infection and analyzed by FACS. The absolute number of donor cells in the spleen was indicated (mean ± SD, n = 4 per group: biological replicates). g WT (Thy1.1 + or Thy1.2 + ), menin KO or rapamycin-treated menin KO OT-1 Tg memory CD8 T cells (Thy1.2 + ) were mixed and transferred into WT congenic mice (Thy1.1 + Thy1.2 + ) as in f . The mice were infected with Lm -OVA the next day and analyzed as in f . The absolute number of donor cells in the spleen is shown (mean ± SD, n = 4 per group: biological replicates). * p

    Journal: Nature Communications

    Article Title: The tumor suppressor menin prevents effector CD8 T-cell dysfunction by targeting mTORC1-dependent metabolic activation

    doi: 10.1038/s41467-018-05854-6

    Figure Lengend Snippet: Rapamycin inhibits dysfunction of menin KO CD8 T cells. a A representative staining profile of CD62L/CD27 on the cell surface of the WT and menin KO effector CD8 T cells on day 7. The percentages of cells are indicated in each quadrant. Naive CD8 T cells were stimulated with anti-TCR-β mAb plus anti-CD28 mAb with IL-2 in the presence or absence of rapamycin for 2 days, and then the cells were further expanded with IL-2 in the absence of rapamycin for an additional 5 days. b Representative staining profiles of CD226 and PD-1 on the surface of the cells in a . c The ELISA for IL-6, IL-10, and OPN in the supernatants of the cells in a restimulated with immobilized anti-TCR-β for 16 h are shown with the standard deviation ( n = 3: biological replicates). d The results of the quantitative RT-PCR analysis of the pro-inflammatory enzymes in the cells in a . The results are presented relative to the expression of Cd3ε mRNA with the standard deviations ( n = 3: technical replicates). e The percentages of SA β-galactosidase (SA β-Gal)-positive cells on day 12 are shown with the standard deviation ( n = 3: biological replicates). f A 1:1 mixture of WT OT-1 Tg effector CD8 T (Thy1.1 + )/ menin KO OT-1 Tg effector CD8 T cells (Thy1.2 + ) or WT (Thy1.1 + )/rapamycin-treated menin KO (Thy1.2 + ) was adoptively transferred into WT congenic (Thy1.1 + Thy1.2 + ) mice. Twenty days after the transfer, the mice were infected with Lm -OVA to activate the donor cells. The donor cells were collected from the spleen on day 5 after Lm -OVA infection and analyzed by FACS. The absolute number of donor cells in the spleen was indicated (mean ± SD, n = 4 per group: biological replicates). g WT (Thy1.1 + or Thy1.2 + ), menin KO or rapamycin-treated menin KO OT-1 Tg memory CD8 T cells (Thy1.2 + ) were mixed and transferred into WT congenic mice (Thy1.1 + Thy1.2 + ) as in f . The mice were infected with Lm -OVA the next day and analyzed as in f . The absolute number of donor cells in the spleen is shown (mean ± SD, n = 4 per group: biological replicates). * p

    Article Snippet: Antibodies used for intracellular and cell-surface staining were as follows: anti-Osteopontin-phycoerythrin (PE) mAb (cat#IC808P; R & D Systems, Minneapolis, MN, USA), anti-IFN-γ-allophycocyanin (APC) mAb (cat#554413; BD Bioscience, San Jose, CA, USA), anti-IFN-γ-fluorescein isothiocyanate (FITC) mAb (cat#554411; BD Bioscience), IL-2-APC mAb (cat#JES6-5H4; TONBO Biosciences, San Diego, CA, USA), anti-Phospho-S6 (Ser235/236)-Alexa Fluor 647 mAb (cat#4851; Cell Signaling Technology, Danvers, MA, USA), anti-Phospho-S6 (Ser240/244) Alexa Fluor 647 mAb (cat#5044; Cell Signaling Technology), anti-S6 ribosomal protein Alexa Fluor 647 mAb (cat#5548; Cell Signaling Technology), anti-CD27-PE mAb (cat#558754; BD Bioscience), anti-CD62L-APC mAb (cat#20-0621; TONBO Biosciences), anti-CD226-APC mAb (cat#128809; BioLegend, San Diego, CA, USA), and anti-PD-1-PE mAb (cat#551892; BD Bioscience).

    Techniques: Staining, Enzyme-linked Immunosorbent Assay, Standard Deviation, Quantitative RT-PCR, Expressing, Mouse Assay, Infection, FACS

    IgM + IgD + CD27 + B cells from blood and spleen share B cell clones with an identical V3-15 CDR3 during a T-independent response Amplification of V3-15-Cμ mRNA sequences was performed from naive and IgM + IgD + CD27 + B cells of a 9-year old child undergoing splenectomy and immunized against Streptococcus pneumoniae and Neisseria meningitidis (plain polysaccharidic vaccines). The following samples were analyzed: blood before immunization, blood at the time of splenectomy (i.e. 8 days after immunization), spleen, blood 5 weeks after immunization. The first V3-15-specific PCR products were further amplified with V3-15-specific FR3 and C7μ primers, and the resulting products fractionated by denaturing gel electrophoresis. A specific CDR3 size was excised from the gel after silver staining and reamplified with the same FR3 and Cμ primers, and sequences determined after cloning. Several PCR amplifications were performed from two independent cDNAs for each cell sample. The recurrent CDR3s encompassing the V3-15 and J H 3 junctions observed in the various IgM + IgD + CD27 + fractions are shown, with the most frequently occurring sequence taken as reference (CDR3 is defined as amino acids included between the conserved Cys residue of FR3 and Trp residue of J H segments). The asterisk (*) marks clones found repeatedly in independent PCR amplifications.

    Journal: Blood

    Article Title: Human blood IgM "memory" B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire

    doi: 10.1182/blood-2004-01-0346

    Figure Lengend Snippet: IgM + IgD + CD27 + B cells from blood and spleen share B cell clones with an identical V3-15 CDR3 during a T-independent response Amplification of V3-15-Cμ mRNA sequences was performed from naive and IgM + IgD + CD27 + B cells of a 9-year old child undergoing splenectomy and immunized against Streptococcus pneumoniae and Neisseria meningitidis (plain polysaccharidic vaccines). The following samples were analyzed: blood before immunization, blood at the time of splenectomy (i.e. 8 days after immunization), spleen, blood 5 weeks after immunization. The first V3-15-specific PCR products were further amplified with V3-15-specific FR3 and C7μ primers, and the resulting products fractionated by denaturing gel electrophoresis. A specific CDR3 size was excised from the gel after silver staining and reamplified with the same FR3 and Cμ primers, and sequences determined after cloning. Several PCR amplifications were performed from two independent cDNAs for each cell sample. The recurrent CDR3s encompassing the V3-15 and J H 3 junctions observed in the various IgM + IgD + CD27 + fractions are shown, with the most frequently occurring sequence taken as reference (CDR3 is defined as amino acids included between the conserved Cys residue of FR3 and Trp residue of J H segments). The asterisk (*) marks clones found repeatedly in independent PCR amplifications.

    Article Snippet: The following antibodies coupled with biotin, fluorescein isothiocyanate (FTTC), R-phycoerythrin (PE), allophycocyanin (APC), Cy-Chrome™ (Cy) or with the tandem dye PE-Cyanin 5.1 (PC5) were used for flow cytometry or cell sorting: PC5-anti-CD19 (clone J4.119) and PE-anti-CD27 (clone 1A4-CD27) from Beckman Coulter (Fullerton, CA); APC-anti-CD19 (clone HIB19), Cy-anti-CD21 (clone B-Ly4), FTTC-anti-CD27 (clone M-T271), PE-anti-CD23 (clone M-L233) and biotin anti-IgD (clone IA6-2) from BD-Pharmingen (San Jose, CA); goat anti-human IgD-FITC and biotinylated goat F(ab′)2 anti-human IgM from Caltag (Burlingame, CA).

    Techniques: Clone Assay, Amplification, Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Silver Staining, Sequencing

    CDlc marks strongly splenic marginal zone B cells in humans Serial cryosections of an adult human spleen are stained with anti-CD20, anti-IgD, anti-CD 1c and anti-CD27 antibodies (ABC technique; Original magnification: 25X) Marginal zone B cells are IgD low CD27 + CDlc high . Note the more intense staining for IgD of the corona (Co) compared to the marginal zone B cells (MZ) while the reverse is true for CDlc. The intense IgD staining of outer marginal zone B cells has been described previousl(16). GC, germinal center.

    Journal: Blood

    Article Title: Human blood IgM "memory" B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire

    doi: 10.1182/blood-2004-01-0346

    Figure Lengend Snippet: CDlc marks strongly splenic marginal zone B cells in humans Serial cryosections of an adult human spleen are stained with anti-CD20, anti-IgD, anti-CD 1c and anti-CD27 antibodies (ABC technique; Original magnification: 25X) Marginal zone B cells are IgD low CD27 + CDlc high . Note the more intense staining for IgD of the corona (Co) compared to the marginal zone B cells (MZ) while the reverse is true for CDlc. The intense IgD staining of outer marginal zone B cells has been described previousl(16). GC, germinal center.

    Article Snippet: The following antibodies coupled with biotin, fluorescein isothiocyanate (FTTC), R-phycoerythrin (PE), allophycocyanin (APC), Cy-Chrome™ (Cy) or with the tandem dye PE-Cyanin 5.1 (PC5) were used for flow cytometry or cell sorting: PC5-anti-CD19 (clone J4.119) and PE-anti-CD27 (clone 1A4-CD27) from Beckman Coulter (Fullerton, CA); APC-anti-CD19 (clone HIB19), Cy-anti-CD21 (clone B-Ly4), FTTC-anti-CD27 (clone M-T271), PE-anti-CD23 (clone M-L233) and biotin anti-IgD (clone IA6-2) from BD-Pharmingen (San Jose, CA); goat anti-human IgD-FITC and biotinylated goat F(ab′)2 anti-human IgM from Caltag (Burlingame, CA).

    Techniques: Staining

    Presence of an IgM + IgD + CD27 + subset in hyper-IgM patients Patients L.D. and C.A. (patient one) have been reported previously (20,21). Control is a eleven-year-old child, age-matched with patient C.A. Since IgD + CD27 + cells co-express IgM, the IgM + IgD + CD27 + subset is analyzed after IgD, CD27 and CD19 labeling of purified B cells. IgD and CD27 expression is shown after gating on CD19-positive cells. Percentages of cells in the naive and the two CD27 + quadrants are indicated.

    Journal: Blood

    Article Title: Human blood IgM "memory" B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire

    doi: 10.1182/blood-2004-01-0346

    Figure Lengend Snippet: Presence of an IgM + IgD + CD27 + subset in hyper-IgM patients Patients L.D. and C.A. (patient one) have been reported previously (20,21). Control is a eleven-year-old child, age-matched with patient C.A. Since IgD + CD27 + cells co-express IgM, the IgM + IgD + CD27 + subset is analyzed after IgD, CD27 and CD19 labeling of purified B cells. IgD and CD27 expression is shown after gating on CD19-positive cells. Percentages of cells in the naive and the two CD27 + quadrants are indicated.

    Article Snippet: The following antibodies coupled with biotin, fluorescein isothiocyanate (FTTC), R-phycoerythrin (PE), allophycocyanin (APC), Cy-Chrome™ (Cy) or with the tandem dye PE-Cyanin 5.1 (PC5) were used for flow cytometry or cell sorting: PC5-anti-CD19 (clone J4.119) and PE-anti-CD27 (clone 1A4-CD27) from Beckman Coulter (Fullerton, CA); APC-anti-CD19 (clone HIB19), Cy-anti-CD21 (clone B-Ly4), FTTC-anti-CD27 (clone M-T271), PE-anti-CD23 (clone M-L233) and biotin anti-IgD (clone IA6-2) from BD-Pharmingen (San Jose, CA); goat anti-human IgD-FITC and biotinylated goat F(ab′)2 anti-human IgM from Caltag (Burlingame, CA).

    Techniques: Labeling, Purification, Expressing

    A common gene expression signature for IgM + IgD + CD27 + B cells from blood and spleen Each column represents microarray data from a sample of the indicated cell subtype and each row represents the expression of a single gene. The spleen IgD + CD27 + and IgD − CD27 + populations are obtained from two separate donors, with one of the two samples prepared in duplicate. Red squares indicate increased expression and green squares indicate decreased expression relative to the median expression of the gene according to the color bar shown. Gray squares indicate missing or excluded data, a) The array dendrogram obtained by clustering the 49 genes that differentiated (p

    Journal: Blood

    Article Title: Human blood IgM "memory" B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire

    doi: 10.1182/blood-2004-01-0346

    Figure Lengend Snippet: A common gene expression signature for IgM + IgD + CD27 + B cells from blood and spleen Each column represents microarray data from a sample of the indicated cell subtype and each row represents the expression of a single gene. The spleen IgD + CD27 + and IgD − CD27 + populations are obtained from two separate donors, with one of the two samples prepared in duplicate. Red squares indicate increased expression and green squares indicate decreased expression relative to the median expression of the gene according to the color bar shown. Gray squares indicate missing or excluded data, a) The array dendrogram obtained by clustering the 49 genes that differentiated (p

    Article Snippet: The following antibodies coupled with biotin, fluorescein isothiocyanate (FTTC), R-phycoerythrin (PE), allophycocyanin (APC), Cy-Chrome™ (Cy) or with the tandem dye PE-Cyanin 5.1 (PC5) were used for flow cytometry or cell sorting: PC5-anti-CD19 (clone J4.119) and PE-anti-CD27 (clone 1A4-CD27) from Beckman Coulter (Fullerton, CA); APC-anti-CD19 (clone HIB19), Cy-anti-CD21 (clone B-Ly4), FTTC-anti-CD27 (clone M-T271), PE-anti-CD23 (clone M-L233) and biotin anti-IgD (clone IA6-2) from BD-Pharmingen (San Jose, CA); goat anti-human IgD-FITC and biotinylated goat F(ab′)2 anti-human IgM from Caltag (Burlingame, CA).

    Techniques: Expressing, Microarray

    Development and diversification of IgM + IgD + CD27 + peripheral B cells from normal and asplenic children A, C. Percentage of IgM + IgD + CD27 + peripheral B cells from normal (A) and asplenic children (C) below five years. B, D. Mutation frequency of rearranged V3-23 genes from IgM + IgD + CD27 + peripheral B cells of normal (B) and asplenic children (D). Each bar represents the mutation frequency of one individual and the values marked above represent the mutation range over the 288 bp V3-23 sequence analyzed. Normal adult values are pooled from five individuals. E. IgD/CD27 staining profiles of peripheral CD19 + lymphocytes of asplenic individuals from 14 months to 71 years. Percentages of cells in each CD27 + quadrants are indicated. A complete description of asplenic patients is given in Supplementary Table.

    Journal: Blood

    Article Title: Human blood IgM "memory" B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire

    doi: 10.1182/blood-2004-01-0346

    Figure Lengend Snippet: Development and diversification of IgM + IgD + CD27 + peripheral B cells from normal and asplenic children A, C. Percentage of IgM + IgD + CD27 + peripheral B cells from normal (A) and asplenic children (C) below five years. B, D. Mutation frequency of rearranged V3-23 genes from IgM + IgD + CD27 + peripheral B cells of normal (B) and asplenic children (D). Each bar represents the mutation frequency of one individual and the values marked above represent the mutation range over the 288 bp V3-23 sequence analyzed. Normal adult values are pooled from five individuals. E. IgD/CD27 staining profiles of peripheral CD19 + lymphocytes of asplenic individuals from 14 months to 71 years. Percentages of cells in each CD27 + quadrants are indicated. A complete description of asplenic patients is given in Supplementary Table.

    Article Snippet: The following antibodies coupled with biotin, fluorescein isothiocyanate (FTTC), R-phycoerythrin (PE), allophycocyanin (APC), Cy-Chrome™ (Cy) or with the tandem dye PE-Cyanin 5.1 (PC5) were used for flow cytometry or cell sorting: PC5-anti-CD19 (clone J4.119) and PE-anti-CD27 (clone 1A4-CD27) from Beckman Coulter (Fullerton, CA); APC-anti-CD19 (clone HIB19), Cy-anti-CD21 (clone B-Ly4), FTTC-anti-CD27 (clone M-T271), PE-anti-CD23 (clone M-L233) and biotin anti-IgD (clone IA6-2) from BD-Pharmingen (San Jose, CA); goat anti-human IgD-FITC and biotinylated goat F(ab′)2 anti-human IgM from Caltag (Burlingame, CA).

    Techniques: Mutagenesis, Sequencing, Staining

    Blood and spleen IgM + IgD + CD27 + subsets share markers specific of marginal zone B cells Purified B cells from blood and spleen are analyzed separately for IgM, CD21, CD23 and CDlc surface expression after gating of the three different CD19-positive lymphocyte subsets distinguished by IgD and CD27 labelling. These data correspond to one representative case out of 4 different individuals. Naive B cells (IgD + CD27 − ), bold line; IgD + CD27 + B cells, grey shadow; IgD − CD27 + , thin line. Percentages of cells in the two CD27 + quadrants are indicated. The absence of IgM-positive cells among the IgD − CD27 + subset is noticeable.

    Journal: Blood

    Article Title: Human blood IgM "memory" B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire

    doi: 10.1182/blood-2004-01-0346

    Figure Lengend Snippet: Blood and spleen IgM + IgD + CD27 + subsets share markers specific of marginal zone B cells Purified B cells from blood and spleen are analyzed separately for IgM, CD21, CD23 and CDlc surface expression after gating of the three different CD19-positive lymphocyte subsets distinguished by IgD and CD27 labelling. These data correspond to one representative case out of 4 different individuals. Naive B cells (IgD + CD27 − ), bold line; IgD + CD27 + B cells, grey shadow; IgD − CD27 + , thin line. Percentages of cells in the two CD27 + quadrants are indicated. The absence of IgM-positive cells among the IgD − CD27 + subset is noticeable.

    Article Snippet: The following antibodies coupled with biotin, fluorescein isothiocyanate (FTTC), R-phycoerythrin (PE), allophycocyanin (APC), Cy-Chrome™ (Cy) or with the tandem dye PE-Cyanin 5.1 (PC5) were used for flow cytometry or cell sorting: PC5-anti-CD19 (clone J4.119) and PE-anti-CD27 (clone 1A4-CD27) from Beckman Coulter (Fullerton, CA); APC-anti-CD19 (clone HIB19), Cy-anti-CD21 (clone B-Ly4), FTTC-anti-CD27 (clone M-T271), PE-anti-CD23 (clone M-L233) and biotin anti-IgD (clone IA6-2) from BD-Pharmingen (San Jose, CA); goat anti-human IgD-FITC and biotinylated goat F(ab′)2 anti-human IgM from Caltag (Burlingame, CA).

    Techniques: Purification, Expressing

    Representative dot plot, showing an increased percentage of CD62L negative expressing cells among the subset of CD8 + CD45RO − CD27 − T cells of the patient. The percentages of positive cells in each quadrant are shown in the right upper corner.

    Journal: British Journal of Cancer

    Article Title: Effector CD8+CD45RO−CD27−T cells have signalling defects in patients with squamous cell carcinoma of the head and neck

    doi: 10.1038/sj.bjc.6600694

    Figure Lengend Snippet: Representative dot plot, showing an increased percentage of CD62L negative expressing cells among the subset of CD8 + CD45RO − CD27 − T cells of the patient. The percentages of positive cells in each quadrant are shown in the right upper corner.

    Article Snippet: Staining for flow cytometry Aliquots of PBMC were stained for flow cytometry, using a panel of labelled monoclonal antibodies (mAbs) specific for cell surface-associated lymphocyte antigens as follows: anti-CD8-PE-Cy5, anti-CD45RO-ECD, anti-CD45RA-PE, anti-CD27-PE, anti-TCRζ -PE, anti-CD62L-FITC (all from Beckman Coulter, Miami, FL, USA) and anti-CD27-FITC (Caltag Laboratories, Burlingame, CA, USA).

    Techniques: Expressing

    Representative flow cytometry dot plot, demonstrating expression of IFN- γ in the effector T cells (CD8 + CD45RO − CD27 − ) of a normal donor and a patient with cancer after 18 h of in vitro stimulation with PMA and ionomycin as described in Materials and Methods. Note that effector T cells expressing IFN- γ after stimulation are CD27 + in NC and CD27-negative in the patient.

    Journal: British Journal of Cancer

    Article Title: Effector CD8+CD45RO−CD27−T cells have signalling defects in patients with squamous cell carcinoma of the head and neck

    doi: 10.1038/sj.bjc.6600694

    Figure Lengend Snippet: Representative flow cytometry dot plot, demonstrating expression of IFN- γ in the effector T cells (CD8 + CD45RO − CD27 − ) of a normal donor and a patient with cancer after 18 h of in vitro stimulation with PMA and ionomycin as described in Materials and Methods. Note that effector T cells expressing IFN- γ after stimulation are CD27 + in NC and CD27-negative in the patient.

    Article Snippet: Staining for flow cytometry Aliquots of PBMC were stained for flow cytometry, using a panel of labelled monoclonal antibodies (mAbs) specific for cell surface-associated lymphocyte antigens as follows: anti-CD8-PE-Cy5, anti-CD45RO-ECD, anti-CD45RA-PE, anti-CD27-PE, anti-TCRζ -PE, anti-CD62L-FITC (all from Beckman Coulter, Miami, FL, USA) and anti-CD27-FITC (Caltag Laboratories, Burlingame, CA, USA).

    Techniques: Flow Cytometry, Cytometry, Expressing, In Vitro

    Representative flow cytometry data, illustrating an increase in the proportion of CD27-negative cells among CD8 + CD45RO − T cells in the patient. The percentages of positive cells in each quadrant are shown in the right upper corner.

    Journal: British Journal of Cancer

    Article Title: Effector CD8+CD45RO−CD27−T cells have signalling defects in patients with squamous cell carcinoma of the head and neck

    doi: 10.1038/sj.bjc.6600694

    Figure Lengend Snippet: Representative flow cytometry data, illustrating an increase in the proportion of CD27-negative cells among CD8 + CD45RO − T cells in the patient. The percentages of positive cells in each quadrant are shown in the right upper corner.

    Article Snippet: Staining for flow cytometry Aliquots of PBMC were stained for flow cytometry, using a panel of labelled monoclonal antibodies (mAbs) specific for cell surface-associated lymphocyte antigens as follows: anti-CD8-PE-Cy5, anti-CD45RO-ECD, anti-CD45RA-PE, anti-CD27-PE, anti-TCRζ -PE, anti-CD62L-FITC (all from Beckman Coulter, Miami, FL, USA) and anti-CD27-FITC (Caltag Laboratories, Burlingame, CA, USA).

    Techniques: Flow Cytometry, Cytometry

    Box and whisker plots showing MFI for TCR-associated ζ chain in effector T cells of patients and NC. The gate was set on CD8 + CD45RO − CD27 − effector T cells. See the legend to Figure 1 for an explanation of the box plot.

    Journal: British Journal of Cancer

    Article Title: Effector CD8+CD45RO−CD27−T cells have signalling defects in patients with squamous cell carcinoma of the head and neck

    doi: 10.1038/sj.bjc.6600694

    Figure Lengend Snippet: Box and whisker plots showing MFI for TCR-associated ζ chain in effector T cells of patients and NC. The gate was set on CD8 + CD45RO − CD27 − effector T cells. See the legend to Figure 1 for an explanation of the box plot.

    Article Snippet: Staining for flow cytometry Aliquots of PBMC were stained for flow cytometry, using a panel of labelled monoclonal antibodies (mAbs) specific for cell surface-associated lymphocyte antigens as follows: anti-CD8-PE-Cy5, anti-CD45RO-ECD, anti-CD45RA-PE, anti-CD27-PE, anti-TCRζ -PE, anti-CD62L-FITC (all from Beckman Coulter, Miami, FL, USA) and anti-CD27-FITC (Caltag Laboratories, Burlingame, CA, USA).

    Techniques: Whisker Assay

    Correlations between age (in years) and the percentages of CD27-negative cells among CD8 + CD45RO − lymphocytes in the peripheral blood of patients and normal controls. The P -value for the significance of differences in the percentage of these lymphocytes between patients and controls has been calculated after adjustment for the age difference between these groups.

    Journal: British Journal of Cancer

    Article Title: Effector CD8+CD45RO−CD27−T cells have signalling defects in patients with squamous cell carcinoma of the head and neck

    doi: 10.1038/sj.bjc.6600694

    Figure Lengend Snippet: Correlations between age (in years) and the percentages of CD27-negative cells among CD8 + CD45RO − lymphocytes in the peripheral blood of patients and normal controls. The P -value for the significance of differences in the percentage of these lymphocytes between patients and controls has been calculated after adjustment for the age difference between these groups.

    Article Snippet: Staining for flow cytometry Aliquots of PBMC were stained for flow cytometry, using a panel of labelled monoclonal antibodies (mAbs) specific for cell surface-associated lymphocyte antigens as follows: anti-CD8-PE-Cy5, anti-CD45RO-ECD, anti-CD45RA-PE, anti-CD27-PE, anti-TCRζ -PE, anti-CD62L-FITC (all from Beckman Coulter, Miami, FL, USA) and anti-CD27-FITC (Caltag Laboratories, Burlingame, CA, USA).

    Techniques:

    Representative flow cytometry histogram, showing MFI of the ζ chain in effector T cells of a patient and an NC. The gate was set on CD8 + CD45RO − CD27 − effector T cells. Note the substantial decrease of ζ MFI in the patient's T cells.

    Journal: British Journal of Cancer

    Article Title: Effector CD8+CD45RO−CD27−T cells have signalling defects in patients with squamous cell carcinoma of the head and neck

    doi: 10.1038/sj.bjc.6600694

    Figure Lengend Snippet: Representative flow cytometry histogram, showing MFI of the ζ chain in effector T cells of a patient and an NC. The gate was set on CD8 + CD45RO − CD27 − effector T cells. Note the substantial decrease of ζ MFI in the patient's T cells.

    Article Snippet: Staining for flow cytometry Aliquots of PBMC were stained for flow cytometry, using a panel of labelled monoclonal antibodies (mAbs) specific for cell surface-associated lymphocyte antigens as follows: anti-CD8-PE-Cy5, anti-CD45RO-ECD, anti-CD45RA-PE, anti-CD27-PE, anti-TCRζ -PE, anti-CD62L-FITC (all from Beckman Coulter, Miami, FL, USA) and anti-CD27-FITC (Caltag Laboratories, Burlingame, CA, USA).

    Techniques: Flow Cytometry, Cytometry

    Infiltration of B cells and iNOS-expressing PCs in H. pylori + patients. ( A ) Representative gating strategy for analysis of LPLs by flow cytometry: 1) gating on lymphocytes by forward (FSC) and side scatter (SSC) properties; 2) exclusion of T cells (CD3 + ) and monocytes (CD14 + ) and gating on CD19 + B cells; 3) detection of PCs (CD20 − CD27 ++ ), mBCs (CD20 + CD27 + ), and naive B cells (CD20 + CD27 − ); 4) detection of iNOS + CD38 ++ PCs; and 5) detection of IgA + PCs within iNOS + CD38 ++ PCs. ( B ) Quantitative analysis of the flow cytometric data of H. pylori –infected patients according to the gating strategy. Values are depicted as median plus interquartile ranges (Mann–Whitney U test).

    Journal: The Journal of Immunology Author Choice

    Article Title: Mucosal Inducible NO Synthase–Producing IgA+ Plasma Cells in Helicobacter pylori–Infected Patients

    doi: 10.4049/jimmunol.1501330

    Figure Lengend Snippet: Infiltration of B cells and iNOS-expressing PCs in H. pylori + patients. ( A ) Representative gating strategy for analysis of LPLs by flow cytometry: 1) gating on lymphocytes by forward (FSC) and side scatter (SSC) properties; 2) exclusion of T cells (CD3 + ) and monocytes (CD14 + ) and gating on CD19 + B cells; 3) detection of PCs (CD20 − CD27 ++ ), mBCs (CD20 + CD27 + ), and naive B cells (CD20 + CD27 − ); 4) detection of iNOS + CD38 ++ PCs; and 5) detection of IgA + PCs within iNOS + CD38 ++ PCs. ( B ) Quantitative analysis of the flow cytometric data of H. pylori –infected patients according to the gating strategy. Values are depicted as median plus interquartile ranges (Mann–Whitney U test).

    Article Snippet: The following fluorochrome-labeled anti-human mAbs were used: anti-CD3–allophycocyanin-H7 (clone SK7; BD), anti-CD14–allophycocyanin-H7 (clone MφP9; BD), anti-CD19–PE-Cy7 (clone SJ25C1; BD), anti-CD19–V500 (clone HIB19; BD), anti-CD19–allophycocyanin (clone HIB19; BD), anti-CD20–Pacific Orange (clone HI47; Invitrogen by Life Technologies), anti-CD20–PerCP (clone 2H7; eBioscience, Frankfurt am Main, Germany), anti-CD27–PE (clone O323; eBioscience), anti-CD38–PerCP-Cy5.5 (clone HIT2; BD), anti-CD38–PE-Cy7 (clone HIT2; BD), anti-iNOS–Alexa Fluor 405 (clone C11; Santa Cruz Biotechnologies, Heidelberg, Germany), anti-IgA–allophycocyanin (clone IS11-8E10; Miltenyi Biotec, Bergisch Gladbach, Germany).

    Techniques: Expressing, Flow Cytometry, Cytometry, Infection, MANN-WHITNEY

    iNOS-expressing B lineage cells produce pro- and anti-inflammatory cytokines. ( A ) Detection of intracellular iNOS (left) or IFN-γ (right) within CD38 − , CD38 + , and CD38 ++ B-LCL cells by flow cytometry. ( B ) Detection of IFN-γ within iNOS + CD38 ++ and iNOS − CD38 ++ B-LCL cells. ( C ) Relative mRNA expression of IFN-γ , TNF-α , and IL-10 in peripheral or mucosal mBCs during H. pylori infection. mBCs (CD19 + CD20 + CD27 + CD38 − CD3 − CD14 − DAPI − ) were isolated from PBMCs or LPLs, respectively, by FACS. The qPCR data were analyzed by the comparative C t method (2 −ΔΔ Ct ). All values are normalized to the expression of the housekeeping gene GAPDH , and the reference gene was IL-6 . ( D ) Detection of intracellular TNF-α and IFN-γ within mucosal iNOS − and iNOS + mBCs (CD19 + CD20 + CD27 + CD38 − CD3 − CD14 − ) or ( E ) PCs (CD19 + CD20 − CD27 ++ CD38 ++ CD3 − CD14 − ) of an H. pylori –infected patient by flow cytometry. Percentages are indicated.

    Journal: The Journal of Immunology Author Choice

    Article Title: Mucosal Inducible NO Synthase–Producing IgA+ Plasma Cells in Helicobacter pylori–Infected Patients

    doi: 10.4049/jimmunol.1501330

    Figure Lengend Snippet: iNOS-expressing B lineage cells produce pro- and anti-inflammatory cytokines. ( A ) Detection of intracellular iNOS (left) or IFN-γ (right) within CD38 − , CD38 + , and CD38 ++ B-LCL cells by flow cytometry. ( B ) Detection of IFN-γ within iNOS + CD38 ++ and iNOS − CD38 ++ B-LCL cells. ( C ) Relative mRNA expression of IFN-γ , TNF-α , and IL-10 in peripheral or mucosal mBCs during H. pylori infection. mBCs (CD19 + CD20 + CD27 + CD38 − CD3 − CD14 − DAPI − ) were isolated from PBMCs or LPLs, respectively, by FACS. The qPCR data were analyzed by the comparative C t method (2 −ΔΔ Ct ). All values are normalized to the expression of the housekeeping gene GAPDH , and the reference gene was IL-6 . ( D ) Detection of intracellular TNF-α and IFN-γ within mucosal iNOS − and iNOS + mBCs (CD19 + CD20 + CD27 + CD38 − CD3 − CD14 − ) or ( E ) PCs (CD19 + CD20 − CD27 ++ CD38 ++ CD3 − CD14 − ) of an H. pylori –infected patient by flow cytometry. Percentages are indicated.

    Article Snippet: The following fluorochrome-labeled anti-human mAbs were used: anti-CD3–allophycocyanin-H7 (clone SK7; BD), anti-CD14–allophycocyanin-H7 (clone MφP9; BD), anti-CD19–PE-Cy7 (clone SJ25C1; BD), anti-CD19–V500 (clone HIB19; BD), anti-CD19–allophycocyanin (clone HIB19; BD), anti-CD20–Pacific Orange (clone HI47; Invitrogen by Life Technologies), anti-CD20–PerCP (clone 2H7; eBioscience, Frankfurt am Main, Germany), anti-CD27–PE (clone O323; eBioscience), anti-CD38–PerCP-Cy5.5 (clone HIT2; BD), anti-CD38–PE-Cy7 (clone HIT2; BD), anti-iNOS–Alexa Fluor 405 (clone C11; Santa Cruz Biotechnologies, Heidelberg, Germany), anti-IgA–allophycocyanin (clone IS11-8E10; Miltenyi Biotec, Bergisch Gladbach, Germany).

    Techniques: Expressing, Flow Cytometry, Cytometry, Infection, Isolation, FACS, Real-time Polymerase Chain Reaction

    Detection of NO-producing PCs in H. pylori –infected patients. ( A ) Representative gating strategy for the analysis of CD19 + CD3 − CD14 − DAPI − PCs of H. pylori– infected patients by flow cytometry: gating on PCs (CD20 − CD27 ++ ) within all CD19 + B cells, and then on CD38 ++ DAF-FM-T + PCs. ( B ) Quantitative analysis of the data of H. pylori + patients ( n = 3). Values are depicted as median plus interquartile ranges.

    Journal: The Journal of Immunology Author Choice

    Article Title: Mucosal Inducible NO Synthase–Producing IgA+ Plasma Cells in Helicobacter pylori–Infected Patients

    doi: 10.4049/jimmunol.1501330

    Figure Lengend Snippet: Detection of NO-producing PCs in H. pylori –infected patients. ( A ) Representative gating strategy for the analysis of CD19 + CD3 − CD14 − DAPI − PCs of H. pylori– infected patients by flow cytometry: gating on PCs (CD20 − CD27 ++ ) within all CD19 + B cells, and then on CD38 ++ DAF-FM-T + PCs. ( B ) Quantitative analysis of the data of H. pylori + patients ( n = 3). Values are depicted as median plus interquartile ranges.

    Article Snippet: The following fluorochrome-labeled anti-human mAbs were used: anti-CD3–allophycocyanin-H7 (clone SK7; BD), anti-CD14–allophycocyanin-H7 (clone MφP9; BD), anti-CD19–PE-Cy7 (clone SJ25C1; BD), anti-CD19–V500 (clone HIB19; BD), anti-CD19–allophycocyanin (clone HIB19; BD), anti-CD20–Pacific Orange (clone HI47; Invitrogen by Life Technologies), anti-CD20–PerCP (clone 2H7; eBioscience, Frankfurt am Main, Germany), anti-CD27–PE (clone O323; eBioscience), anti-CD38–PerCP-Cy5.5 (clone HIT2; BD), anti-CD38–PE-Cy7 (clone HIT2; BD), anti-iNOS–Alexa Fluor 405 (clone C11; Santa Cruz Biotechnologies, Heidelberg, Germany), anti-IgA–allophycocyanin (clone IS11-8E10; Miltenyi Biotec, Bergisch Gladbach, Germany).

    Techniques: Infection, Flow Cytometry, Cytometry

    somatic hypermutation (SHM) results on the VH3-23 region of IgM on CD19+CD27+ isolated B cells. ( A ) Frequency of mutations on IgM+CD27+ B cells purified from controls and patients. Dots represent results for each subject. Controls: Black dots, all 10 clones mutated and white dots, 9/10 clones mutated. Patients: P1, * and P2, #; ( B ) Nucleotide substitution pattern for P1 (1st evaluation), P2, and control. The same pattern was observed in P1 in the 2nd evaluation. ( C ) The numbers of mutated clones from all studied clones is shown, as well as the frequency of mutations.

    Journal: Journal of Clinical Medicine

    Article Title: Clinical, Immunological, and Functional Characterization of Six Patients with Very High IgM Levels

    doi: 10.3390/jcm9030818

    Figure Lengend Snippet: somatic hypermutation (SHM) results on the VH3-23 region of IgM on CD19+CD27+ isolated B cells. ( A ) Frequency of mutations on IgM+CD27+ B cells purified from controls and patients. Dots represent results for each subject. Controls: Black dots, all 10 clones mutated and white dots, 9/10 clones mutated. Patients: P1, * and P2, #; ( B ) Nucleotide substitution pattern for P1 (1st evaluation), P2, and control. The same pattern was observed in P1 in the 2nd evaluation. ( C ) The numbers of mutated clones from all studied clones is shown, as well as the frequency of mutations.

    Article Snippet: PBMCs were isolated by flow cytometry using FITC-anti-CD19 and PE-anti-CD27 mAb (Invitrogen, Carlsbad, CA, USA).

    Techniques: Isolation, Purification, Clone Assay

    B-cell immunophenotyping. Representative flow cytometric plot showing total CD19+ cells (left panels), memory (CD19+ CD27+IgM+) and switched memory (CD19+ CD27+IgM-) B-cell populations in patients P1 and P2 (right panels), expressed as percentage of total lymphocyte.

    Journal: Journal of Clinical Medicine

    Article Title: Clinical, Immunological, and Functional Characterization of Six Patients with Very High IgM Levels

    doi: 10.3390/jcm9030818

    Figure Lengend Snippet: B-cell immunophenotyping. Representative flow cytometric plot showing total CD19+ cells (left panels), memory (CD19+ CD27+IgM+) and switched memory (CD19+ CD27+IgM-) B-cell populations in patients P1 and P2 (right panels), expressed as percentage of total lymphocyte.

    Article Snippet: PBMCs were isolated by flow cytometry using FITC-anti-CD19 and PE-anti-CD27 mAb (Invitrogen, Carlsbad, CA, USA).

    Techniques:

    CD81 engagement induces the preferential expansion of naïve B lymphocytes. Purified naive (CD27 - ) or memory (CD27 + ) B cells were incubated with complete medium (Control), MG81 and N81 mAbs (5 μg/ml each) (CD81), or SAC (1:30,000, vol/vol).

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

    Article Title: Activation of na?ve B lymphocytes via CD81, a pathogenetic mechanism for hepatitis C virus-associated B lymphocyte disorders

    doi: 10.1073/pnas.0509402102

    Figure Lengend Snippet: CD81 engagement induces the preferential expansion of naïve B lymphocytes. Purified naive (CD27 - ) or memory (CD27 + ) B cells were incubated with complete medium (Control), MG81 and N81 mAbs (5 μg/ml each) (CD81), or SAC (1:30,000, vol/vol).

    Article Snippet: Purified B lymphocytes were stained with phycoerythrin (PE)-anti-CD27 mAb (clone L-128, BD Biosciences), and naïve (CD27- ) and memory (CD27+ ) subsets were sorted by FACSVantage SE (Becton Dickinson) with > 96% purity.

    Techniques: Purification, Incubation

    EBV-infected cells in the periphery of AIM patients are CD27 positive. PBMCs were stained for expression of CD20, a pan-B-cell marker, and CD27, a memory B-cell marker (upper panel). The naive and memory B cells were then sorted by FACS and reanalyzed for purity (middle panels). The purified populations were then tested for the presence of the virus by limiting dilution DNA PCR. In this technique, serial dilutions of each population are prepared and then multiple aliquots of each dilution are tested for the virus by DNA PCR. The PCR products are separated on a gel and detected by Southern blotting.

    Journal: Journal of Virology

    Article Title: Acute Infection with Epstein-Barr Virus Targets and Overwhelms the Peripheral Memory B-Cell Compartment with Resting, Latently Infected Cells

    doi: 10.1128/JVI.78.10.5194-5204.2004

    Figure Lengend Snippet: EBV-infected cells in the periphery of AIM patients are CD27 positive. PBMCs were stained for expression of CD20, a pan-B-cell marker, and CD27, a memory B-cell marker (upper panel). The naive and memory B cells were then sorted by FACS and reanalyzed for purity (middle panels). The purified populations were then tested for the presence of the virus by limiting dilution DNA PCR. In this technique, serial dilutions of each population are prepared and then multiple aliquots of each dilution are tested for the virus by DNA PCR. The PCR products are separated on a gel and detected by Southern blotting.

    Article Snippet: The antibodies and dilutions used were as follows: anti-CD20 fluorescein isothiocyanate (FITC) (DAKO), 1:200; anti-CD5 phycoerythrin (PE) (Pharmingen), 1:200; anti-CD20 Cy5 (DAKO), 1:100; anti-CD27 PE (Pharmingen), 1:100; anti-IgD PE (Southern Biotec), 1:2,000; anti-IgG, -IgA, and -IgM FITC (Jackson), 1:125.

    Techniques: Infection, Staining, Expressing, Marker, FACS, Purification, Polymerase Chain Reaction, Southern Blot

    Gene array comparisons of CD21 + and CD21 –/low CD27 - B cells from pSS patients using the Illumina Human Whole Genome Array

    Journal: Arthritis and rheumatism

    Article Title: Expansion of autoreactive unresponsive CD21-/low B cells in Sjögren's syndrome associated lymphoproliferation

    doi: 10.1002/art.37828

    Figure Lengend Snippet: Gene array comparisons of CD21 + and CD21 –/low CD27 - B cells from pSS patients using the Illumina Human Whole Genome Array

    Article Snippet: For gene expression profile and single cell sorting, PBMCs were sorted by staining the cells with monoclonal anti-human antibodies against CD45-VioBlue, anti-CD27-PE (Miltenyi Biotec), CD21-FITC, CD19-ECD, and CD10-APC (Beckman Coulter).

    Techniques:

    CD27 - CD21 -/low B cells reveal defects in BCR and CD40-mediated activation

    Journal: Arthritis and rheumatism

    Article Title: Expansion of autoreactive unresponsive CD21-/low B cells in Sjögren's syndrome associated lymphoproliferation

    doi: 10.1002/art.37828

    Figure Lengend Snippet: CD27 - CD21 -/low B cells reveal defects in BCR and CD40-mediated activation

    Article Snippet: For gene expression profile and single cell sorting, PBMCs were sorted by staining the cells with monoclonal anti-human antibodies against CD45-VioBlue, anti-CD27-PE (Miltenyi Biotec), CD21-FITC, CD19-ECD, and CD10-APC (Beckman Coulter).

    Techniques: Activation Assay

    FACS analysis of proteins differentially expressed between CD21 + and CD21 –/low CD27 - B cells from pSS patients

    Journal: Arthritis and rheumatism

    Article Title: Expansion of autoreactive unresponsive CD21-/low B cells in Sjögren's syndrome associated lymphoproliferation

    doi: 10.1002/art.37828

    Figure Lengend Snippet: FACS analysis of proteins differentially expressed between CD21 + and CD21 –/low CD27 - B cells from pSS patients

    Article Snippet: For gene expression profile and single cell sorting, PBMCs were sorted by staining the cells with monoclonal anti-human antibodies against CD45-VioBlue, anti-CD27-PE (Miltenyi Biotec), CD21-FITC, CD19-ECD, and CD10-APC (Beckman Coulter).

    Techniques: FACS

    Expansion of an unusual CD27 - CD21 -/low B cell population in pSS patients

    Journal: Arthritis and rheumatism

    Article Title: Expansion of autoreactive unresponsive CD21-/low B cells in Sjögren's syndrome associated lymphoproliferation

    doi: 10.1002/art.37828

    Figure Lengend Snippet: Expansion of an unusual CD27 - CD21 -/low B cell population in pSS patients

    Article Snippet: For gene expression profile and single cell sorting, PBMCs were sorted by staining the cells with monoclonal anti-human antibodies against CD45-VioBlue, anti-CD27-PE (Miltenyi Biotec), CD21-FITC, CD19-ECD, and CD10-APC (Beckman Coulter).

    Techniques:

    HIV-1 infected patients present with expanded populations of blood CD27 − IgA + and CD27 − IgG + B-cells and show inverse patterns of SHM. The percentage of CD27 − IgA + (A) and CD27 − IgG + (B) among total IgA/G expressing B-cells is significantly expanded in HIV-1 infected patients (right panel) as compared with healthy controls (left panel). (C) The number of somatic hypermutations in the VH region of mRNA transcripts of CD27 − B cells (left panel) is increased in HIV-1 infected patients (black bars), as compared with healthy controls (white bars), while an opposite trend is shown for CD27 + B cells (right panel), where the number of somatic hypermutations in the VH region of mRNA transcripts is decreased in HIV-1 infected patients (black bars) as compared with healthy controls (white bars). The number below the bars indicates the number of clones analysed.

    Journal: PLoS ONE

    Article Title: CD27− B-Cells Produce Class Switched and Somatically Hyper-Mutated Antibodies during Chronic HIV-1 Infection

    doi: 10.1371/journal.pone.0005427

    Figure Lengend Snippet: HIV-1 infected patients present with expanded populations of blood CD27 − IgA + and CD27 − IgG + B-cells and show inverse patterns of SHM. The percentage of CD27 − IgA + (A) and CD27 − IgG + (B) among total IgA/G expressing B-cells is significantly expanded in HIV-1 infected patients (right panel) as compared with healthy controls (left panel). (C) The number of somatic hypermutations in the VH region of mRNA transcripts of CD27 − B cells (left panel) is increased in HIV-1 infected patients (black bars), as compared with healthy controls (white bars), while an opposite trend is shown for CD27 + B cells (right panel), where the number of somatic hypermutations in the VH region of mRNA transcripts is decreased in HIV-1 infected patients (black bars) as compared with healthy controls (white bars). The number below the bars indicates the number of clones analysed.

    Article Snippet: Flow cytometric cell-phenotyping on fresh isolated PBMCs were performed using the following fluorochrome conjugated anti-human Abs: anti-IgA-fluorescein isothiocyanate (FITC) (Dako, Stockholm, Sweden), anti-IgG-FITC (Pharmingen, San Diego, CA), anti-CD27-phycoerythrin (PE) (Pharmingen, San Diego, CA) and anti CD19- Cy-Chrome (Cy5) (Pharmingen, San Diego, CA) in addition to isotype control Abs (Pharmingen, San Diego, CA) to set the background staining intensity.

    Techniques: Infection, Expressing, Clone Assay

    AID expression in HIV-1 infected patients and healthy controls. (A) The ex-vivo levels of AID mRNA expression (left panel) are higher in patients (black bars) as compared with controls (white bars) but they reach similar levels upon in vitro stimulation (middle and right panels). The baseline levels of AID mRNA expression correlate with the percentage of CD27 + B cells in healthy controls (B) while there is no correlation for HIV-1 infected patients (C). The anti-CD40 stimulation was performed with an anti-CD40 mAb, IL4 and IL10 while TLR9 stimulation with CpG.

    Journal: PLoS ONE

    Article Title: CD27− B-Cells Produce Class Switched and Somatically Hyper-Mutated Antibodies during Chronic HIV-1 Infection

    doi: 10.1371/journal.pone.0005427

    Figure Lengend Snippet: AID expression in HIV-1 infected patients and healthy controls. (A) The ex-vivo levels of AID mRNA expression (left panel) are higher in patients (black bars) as compared with controls (white bars) but they reach similar levels upon in vitro stimulation (middle and right panels). The baseline levels of AID mRNA expression correlate with the percentage of CD27 + B cells in healthy controls (B) while there is no correlation for HIV-1 infected patients (C). The anti-CD40 stimulation was performed with an anti-CD40 mAb, IL4 and IL10 while TLR9 stimulation with CpG.

    Article Snippet: Flow cytometric cell-phenotyping on fresh isolated PBMCs were performed using the following fluorochrome conjugated anti-human Abs: anti-IgA-fluorescein isothiocyanate (FITC) (Dako, Stockholm, Sweden), anti-IgG-FITC (Pharmingen, San Diego, CA), anti-CD27-phycoerythrin (PE) (Pharmingen, San Diego, CA) and anti CD19- Cy-Chrome (Cy5) (Pharmingen, San Diego, CA) in addition to isotype control Abs (Pharmingen, San Diego, CA) to set the background staining intensity.

    Techniques: Expressing, Infection, Ex Vivo, In Vitro

    The percentages of IgG + IgA + CD27 + switch memory B cells increase after vaccination in young but not in elderly subjects

    Journal: Vaccine

    Article Title: Intrinsic defects in B cell response to seasonal influenza vaccination in elderly humans

    doi: 10.1016/j.vaccine.2010.10.023

    Figure Lengend Snippet: The percentages of IgG + IgA + CD27 + switch memory B cells increase after vaccination in young but not in elderly subjects

    Article Snippet: To detect naive B cells (IgG− /IgA− /CD27− ), IgM memory B cells (IgG− /IgA− /CD27+ ), and switch memory B cells (IgG+ /IgA+ ), blood cells were stained with PE-conjugated anti-CD27 (BD Pharmingen 555441), biotin-SP-conjugated AffiniPure F(ab′)2 fragment anti-human IgG (Jackson ImmunoResearch Laboratories.109-066-098), and biotin-SP-conjugated AffiniPure F(ab′)2 fragment anti-human IgA (Jackson ImmunoResearch Laboratories no.109-066-011).

    Techniques: