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<t>CD4</t> T cell subsets repartition and contribution to the pool of infected cells. A : Monocytes, activated and resting CD4 T-cell (CD4 T Ly) contributions to the pool of infected <t>PBMCs</t> were calculated with the infection level and frequency of each subset. Only significant p values are shown. B : The repartition of resting CD4 T-cell subsets was assessed in twelve acutely HIV-infected individuals (grey) and in ten uninfected individuals (white). The analyzed resting CD4+ subsets are: naive (TN, CD45RA+CCR7+CD27+), central-memory (TCM, CD45RA-CCR7+CD27+), transitional-memory (TTM, CD45RA−CCR7−CD27+) and effector-memory cells (TEM, CD45RA−CCR7−CD27−). Results are expressed as the percentage of resting CD4 T cells. C : Resting CD4 T-cell subset contributions to the pool of infected resting CD4 T cells were calculated with the infection level and frequency of each subset.
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Images

1) Product Images from "A Single HIV-1 Cluster and a Skewed Immune Homeostasis Drive the Early Spread of HIV among Resting CD4+ Cell Subsets within One Month Post-Infection"

Article Title: A Single HIV-1 Cluster and a Skewed Immune Homeostasis Drive the Early Spread of HIV among Resting CD4+ Cell Subsets within One Month Post-Infection

Journal: PLoS ONE

doi: 10.1371/journal.pone.0064219

CD4 T cell subsets repartition and contribution to the pool of infected cells. A : Monocytes, activated and resting CD4 T-cell (CD4 T Ly) contributions to the pool of infected PBMCs were calculated with the infection level and frequency of each subset. Only significant p values are shown. B : The repartition of resting CD4 T-cell subsets was assessed in twelve acutely HIV-infected individuals (grey) and in ten uninfected individuals (white). The analyzed resting CD4+ subsets are: naive (TN, CD45RA+CCR7+CD27+), central-memory (TCM, CD45RA-CCR7+CD27+), transitional-memory (TTM, CD45RA−CCR7−CD27+) and effector-memory cells (TEM, CD45RA−CCR7−CD27−). Results are expressed as the percentage of resting CD4 T cells. C : Resting CD4 T-cell subset contributions to the pool of infected resting CD4 T cells were calculated with the infection level and frequency of each subset.
Figure Legend Snippet: CD4 T cell subsets repartition and contribution to the pool of infected cells. A : Monocytes, activated and resting CD4 T-cell (CD4 T Ly) contributions to the pool of infected PBMCs were calculated with the infection level and frequency of each subset. Only significant p values are shown. B : The repartition of resting CD4 T-cell subsets was assessed in twelve acutely HIV-infected individuals (grey) and in ten uninfected individuals (white). The analyzed resting CD4+ subsets are: naive (TN, CD45RA+CCR7+CD27+), central-memory (TCM, CD45RA-CCR7+CD27+), transitional-memory (TTM, CD45RA−CCR7−CD27+) and effector-memory cells (TEM, CD45RA−CCR7−CD27−). Results are expressed as the percentage of resting CD4 T cells. C : Resting CD4 T-cell subset contributions to the pool of infected resting CD4 T cells were calculated with the infection level and frequency of each subset.

Techniques Used: Infection, Transmission Electron Microscopy

2) Product Images from "Blocking TLR7- and TLR9-mediated IFN-? Production by Plasmacytoid Dendritic Cells Does Not Diminish Immune Activation in Early SIV Infection"

Article Title: Blocking TLR7- and TLR9-mediated IFN-? Production by Plasmacytoid Dendritic Cells Does Not Diminish Immune Activation in Early SIV Infection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003530

TLR7 and TLR9 antagonist selectively blocks proinflammatory cytokine production from uninfected rhesus macaque pDC. (A) Representative gating strategy to define pDC, mDC, monocytes (Mo) and macrophages (Mø) by flow cytometry in rhesus macaque PBMC and lymph node. The lymph node gate to define DC includes autofluorescent cells that encroach on the Lineage+ subset, as previously described [23] . (B) PBMC and lymph node cell suspensions from SIV-naive animals were stimulated with iSIV or influenza virus with and without DV056 and intracellular expression of IFN-α and TNF-α in pDC was determined by flow cytometry. Positive labeling with cytokine-specific antibody as shown by the boxed areas was based on isotype control antibody. Numbers represent the percent positive cells. (C) PBMC, pDC-depleted PBMC and PBMC pretreated with DV056 were stimulated with iSIV, live SIV, influenza virus or CpG-C and supernatants were analyzed for IFN-α by ELISA. Unstimulated PBMC served as a negative control. (D) PBMC and lymph node cell suspensions were stimulated with iSIV or influenza virus and IFN-α and/or TNF-α in mDC, monocytes or macrophages were determined by flow cytometry. SSC = side scatter.
Figure Legend Snippet: TLR7 and TLR9 antagonist selectively blocks proinflammatory cytokine production from uninfected rhesus macaque pDC. (A) Representative gating strategy to define pDC, mDC, monocytes (Mo) and macrophages (Mø) by flow cytometry in rhesus macaque PBMC and lymph node. The lymph node gate to define DC includes autofluorescent cells that encroach on the Lineage+ subset, as previously described [23] . (B) PBMC and lymph node cell suspensions from SIV-naive animals were stimulated with iSIV or influenza virus with and without DV056 and intracellular expression of IFN-α and TNF-α in pDC was determined by flow cytometry. Positive labeling with cytokine-specific antibody as shown by the boxed areas was based on isotype control antibody. Numbers represent the percent positive cells. (C) PBMC, pDC-depleted PBMC and PBMC pretreated with DV056 were stimulated with iSIV, live SIV, influenza virus or CpG-C and supernatants were analyzed for IFN-α by ELISA. Unstimulated PBMC served as a negative control. (D) PBMC and lymph node cell suspensions were stimulated with iSIV or influenza virus and IFN-α and/or TNF-α in mDC, monocytes or macrophages were determined by flow cytometry. SSC = side scatter.

Techniques Used: Flow Cytometry, Cytometry, Expressing, Labeling, Enzyme-linked Immunosorbent Assay, Negative Control

3) Product Images from "Dysregulation of Th17 Cells during the Early Post-Transplant Period in Patients under Calcineurin Inhibitor Based Immunosuppression"

Article Title: Dysregulation of Th17 Cells during the Early Post-Transplant Period in Patients under Calcineurin Inhibitor Based Immunosuppression

Journal: PLoS ONE

doi: 10.1371/journal.pone.0042011

Distribution of T naïve , T CM , T EM subpopulations and IL-17 + /T naïve , IL-17 + /T EM and IL-17 + /T CM , subpopulations of CD4 + T lymphocytes at 1 and 3 month after transplantation compared to before transplantation. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of GolgiStop.CD4 + lymphocytes were stained with mAbs to CD45RA and CCR7, which identified three subsets. In addition, analysis of IL-17 in CD4 + T cell subsets by intracellular flow cytometry was done. After surface staining with CD45 and CCR7 mAbs, cells were fixated and permeabilized and intracellular accumulated cytokines were detected with IL-17 mAbs. (A) T naïve /CD4 + T (CD45RA + CCR7 + /CD4 + Tcells), (B) IL-17 + /T naïve , (C) T CM /CD4 + T (CD45RA − CCR7 + /CD4 + Tcells), (D) IL-17 + /T CM + , (E) T EM /CD4 + T (CD45RA − CCR7 − /CD4 + Tcells), (F) IL-17 + /T EM + . Bars show the means. * P
Figure Legend Snippet: Distribution of T naïve , T CM , T EM subpopulations and IL-17 + /T naïve , IL-17 + /T EM and IL-17 + /T CM , subpopulations of CD4 + T lymphocytes at 1 and 3 month after transplantation compared to before transplantation. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of GolgiStop.CD4 + lymphocytes were stained with mAbs to CD45RA and CCR7, which identified three subsets. In addition, analysis of IL-17 in CD4 + T cell subsets by intracellular flow cytometry was done. After surface staining with CD45 and CCR7 mAbs, cells were fixated and permeabilized and intracellular accumulated cytokines were detected with IL-17 mAbs. (A) T naïve /CD4 + T (CD45RA + CCR7 + /CD4 + Tcells), (B) IL-17 + /T naïve , (C) T CM /CD4 + T (CD45RA − CCR7 + /CD4 + Tcells), (D) IL-17 + /T CM + , (E) T EM /CD4 + T (CD45RA − CCR7 − /CD4 + Tcells), (F) IL-17 + /T EM + . Bars show the means. * P

Techniques Used: Transplantation Assay, Ex Vivo, Staining, Flow Cytometry, Cytometry

Expression of IL-1beta, and HMGB1 associated with Th17 cell at 1 and 3 month after transplantation compared to before transplantation. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of GolgiStop. PBMC from all groups were treated as described in Figure 1 and Materials and Methods . The expression of IL-1beta (A), HMGB1(B) mRNA was measured using real-time PCR. Bars show the means.
Figure Legend Snippet: Expression of IL-1beta, and HMGB1 associated with Th17 cell at 1 and 3 month after transplantation compared to before transplantation. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of GolgiStop. PBMC from all groups were treated as described in Figure 1 and Materials and Methods . The expression of IL-1beta (A), HMGB1(B) mRNA was measured using real-time PCR. Bars show the means.

Techniques Used: Expressing, Transplantation Assay, Ex Vivo, Real-time Polymerase Chain Reaction

Distribution of lymphocyte and CD4+ T cell subtype at 1 and 3 month after transplantation compared to before transplantation. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of Golgi Stop. The percentage of Target cells was measured by flowcytometry. The frequency (%) of Lymphocyte/Leukocyte cells (A), CD4 + T/Lymphocyte cells (B), IFN-γ + /CD4 + T cells (C), IL-4 + /CD4 + T cells (D) CD25 + FOXP3 + /CD4 + T cells (E) and IL-17 + /CD4 + T cells (F) in patients before KT, patients at 1month after KT and patients at 3 month after KT. Bars show the means. * P
Figure Legend Snippet: Distribution of lymphocyte and CD4+ T cell subtype at 1 and 3 month after transplantation compared to before transplantation. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of Golgi Stop. The percentage of Target cells was measured by flowcytometry. The frequency (%) of Lymphocyte/Leukocyte cells (A), CD4 + T/Lymphocyte cells (B), IFN-γ + /CD4 + T cells (C), IL-4 + /CD4 + T cells (D) CD25 + FOXP3 + /CD4 + T cells (E) and IL-17 + /CD4 + T cells (F) in patients before KT, patients at 1month after KT and patients at 3 month after KT. Bars show the means. * P

Techniques Used: Transplantation Assay, Ex Vivo

Effects of Tac in Th1, Th2, Th17 and Treg subpopulations of CD4+T lymphocytes from the peripheral blood of healthy donors. PBMC were preincubated for 1 h in the presence of Tac and stimulated with 1 µg/ml anti-CD3 and anti-CD28. Flow cytometry of intracellular IFN-r (A), IL-4 (B), Th17 (C) and Treg (D) in CD4+ T cells stimulated in the presence of plate-bound anti-CD3 plus anti-CD28, assessed after 48 h and then stimulated for 4 h with PMA and ionomycin in the presence of GolgiStop. The data are representative of three independent experiments. The values are expressed as the mean ± SEM. * P
Figure Legend Snippet: Effects of Tac in Th1, Th2, Th17 and Treg subpopulations of CD4+T lymphocytes from the peripheral blood of healthy donors. PBMC were preincubated for 1 h in the presence of Tac and stimulated with 1 µg/ml anti-CD3 and anti-CD28. Flow cytometry of intracellular IFN-r (A), IL-4 (B), Th17 (C) and Treg (D) in CD4+ T cells stimulated in the presence of plate-bound anti-CD3 plus anti-CD28, assessed after 48 h and then stimulated for 4 h with PMA and ionomycin in the presence of GolgiStop. The data are representative of three independent experiments. The values are expressed as the mean ± SEM. * P

Techniques Used: Flow Cytometry, Cytometry

Effect of Tac in Th17 subpopulations of CD4+T lymphocytes from the peripheral blood of early-post transplant recipients. We used flow cytometry to examine how Tacrolimus regulates in vitro Th17 subpopulations of CD4+ T lymphocytes in Th17-polarizing condition. PBMC from renal transplant recipients were preincubated for 1 h in the presence of Tacrolimus and stimulated with Th17-polarizing condition for 48 h. Anti-CD3 (1 µg/ml), anti-CD28 (1 µg/ml), IL-1b (20 ng/ml), IL-6 (20 ng/ml) and IL-23 (20 ng/ml) were added to stimulate the differentiation of Th17 cells. Neutralizing antibodies to IFN-gamma (2 µg/ml) and IL-4 (2 µg/ml) were added in some experiments (R D Systems). Flow cytometry of intracellular Th17 in CD4+ T cells stimulated in the presence of Th17-polarizing condition, assessed after 48 h and then stimulated for 4 h with PMA and ionomycin in the presence of GolgiStop. The data are representative of three independent experiments. The values are expressed as the mean ± SEM.
Figure Legend Snippet: Effect of Tac in Th17 subpopulations of CD4+T lymphocytes from the peripheral blood of early-post transplant recipients. We used flow cytometry to examine how Tacrolimus regulates in vitro Th17 subpopulations of CD4+ T lymphocytes in Th17-polarizing condition. PBMC from renal transplant recipients were preincubated for 1 h in the presence of Tacrolimus and stimulated with Th17-polarizing condition for 48 h. Anti-CD3 (1 µg/ml), anti-CD28 (1 µg/ml), IL-1b (20 ng/ml), IL-6 (20 ng/ml) and IL-23 (20 ng/ml) were added to stimulate the differentiation of Th17 cells. Neutralizing antibodies to IFN-gamma (2 µg/ml) and IL-4 (2 µg/ml) were added in some experiments (R D Systems). Flow cytometry of intracellular Th17 in CD4+ T cells stimulated in the presence of Th17-polarizing condition, assessed after 48 h and then stimulated for 4 h with PMA and ionomycin in the presence of GolgiStop. The data are representative of three independent experiments. The values are expressed as the mean ± SEM.

Techniques Used: Flow Cytometry, Cytometry, In Vitro

Flow cytometric analysis of T cell subsets. PBMCs were stained with anti-CD4 PE-cy7, anti-CD25 APC, anti-IFN-γ FITC, anti-IL-17 PE, anti-IL-4 APC and anti-Foxp3 FITC. CD4+ cells were gated for further analysis. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of Golgi Stop. The percentage of Target cells was measured by flowcytometry. The frequency (%) of Lymphocyte/Leukocyte cells, CD4 + T/Lymphocyte cells, IL-17+/CD4 + T cells, IFN-γ + /CD4 + T cells, IL-4 + /CD4 + T cells (A) and CD25 + FOXP3 + /CD4 + T cells (B) in patients before KT, patients at 1month after KT and patients at 3 month after KT. After surface staining with anti-CD4, CD45 and CCR7 mAbs, cells were fixated and permeabilized and intracellular accumulated cytokines were detected with IL-17 mAbs. T naïve /CD4 + T (CD45RA + CCR7 + /CD4 + Tcells), IL-17 + /T naïve , T CM /CD4 + T (CD45RA − CCR7 + /CD4 + Tcells), IL-17 + /T CM + and T EM /CD4 + T (CD45RA − CCR7 − /CD4 + Tcells), IL-17 + /T EM + (C).
Figure Legend Snippet: Flow cytometric analysis of T cell subsets. PBMCs were stained with anti-CD4 PE-cy7, anti-CD25 APC, anti-IFN-γ FITC, anti-IL-17 PE, anti-IL-4 APC and anti-Foxp3 FITC. CD4+ cells were gated for further analysis. PBMC from patients before KT, patients at 1month after KT and patients at 3 month after KT were stimulated for 4 h ex vivo with PMA and ionomycin in the presence of Golgi Stop. The percentage of Target cells was measured by flowcytometry. The frequency (%) of Lymphocyte/Leukocyte cells, CD4 + T/Lymphocyte cells, IL-17+/CD4 + T cells, IFN-γ + /CD4 + T cells, IL-4 + /CD4 + T cells (A) and CD25 + FOXP3 + /CD4 + T cells (B) in patients before KT, patients at 1month after KT and patients at 3 month after KT. After surface staining with anti-CD4, CD45 and CCR7 mAbs, cells were fixated and permeabilized and intracellular accumulated cytokines were detected with IL-17 mAbs. T naïve /CD4 + T (CD45RA + CCR7 + /CD4 + Tcells), IL-17 + /T naïve , T CM /CD4 + T (CD45RA − CCR7 + /CD4 + Tcells), IL-17 + /T CM + and T EM /CD4 + T (CD45RA − CCR7 − /CD4 + Tcells), IL-17 + /T EM + (C).

Techniques Used: Flow Cytometry, Staining, Ex Vivo

4) Product Images from "Progressive contraction of the latent HIV reservoir around a core of less-differentiated CD4+ memory T Cells"

Article Title: Progressive contraction of the latent HIV reservoir around a core of less-differentiated CD4+ memory T Cells

Journal: Nature Communications

doi: 10.1038/ncomms6407

During HIV infection the relative size of the T SCM subset within memory CD4 T cells remains stable. ( a ) The gating strategy used to sort the memory cell subsets: CD4-enriched PBMC were stained with a cocktail of antibodies (see Methods) and doublets were excluded on the basis of both forward scatter (FSC) and side scatter (SSC). Resting CD4 + T cells were gated after exclusion of CD19 + , CD14 + , CD8 + , HLADR + , CD69 + , CD25 + cells. Resting T SCM were sorted on the basis of the following phenotype: CD45RA + CD45RO − CCR7 + CD62L + CD27 + CD95 + . CXCR3 and CD122 expression by T SCM is also shown. ( b ) The absolute number of cells in each memory subset (T SCM , T CM , T EM and T IM ) was determined in HIV-infected patients with undetectable plasma viral load on ART and with CD4 cell counts above 500 per mm 3 , who were tested for integrated virus (UND, n =38), as well as in age- and sex-matched viremic patients (VIR, n =18) and HIV-seronegative healthy donors (HD, n =20). The Kruskal–Wallis and Dunn tests were used for statistical analysis (* P
Figure Legend Snippet: During HIV infection the relative size of the T SCM subset within memory CD4 T cells remains stable. ( a ) The gating strategy used to sort the memory cell subsets: CD4-enriched PBMC were stained with a cocktail of antibodies (see Methods) and doublets were excluded on the basis of both forward scatter (FSC) and side scatter (SSC). Resting CD4 + T cells were gated after exclusion of CD19 + , CD14 + , CD8 + , HLADR + , CD69 + , CD25 + cells. Resting T SCM were sorted on the basis of the following phenotype: CD45RA + CD45RO − CCR7 + CD62L + CD27 + CD95 + . CXCR3 and CD122 expression by T SCM is also shown. ( b ) The absolute number of cells in each memory subset (T SCM , T CM , T EM and T IM ) was determined in HIV-infected patients with undetectable plasma viral load on ART and with CD4 cell counts above 500 per mm 3 , who were tested for integrated virus (UND, n =38), as well as in age- and sex-matched viremic patients (VIR, n =18) and HIV-seronegative healthy donors (HD, n =20). The Kruskal–Wallis and Dunn tests were used for statistical analysis (* P

Techniques Used: Infection, Staining, Expressing

5) Product Images from "Early short-term treatment with neutralizing human monoclonal antibodies halts SHIV infection in newborn macaques"

Article Title: Early short-term treatment with neutralizing human monoclonal antibodies halts SHIV infection in newborn macaques

Journal: Nature medicine

doi: 10.1038/nm.4063

SHIV SF162P3 -associated viremia is not established in plasma or PBMC of NmAb-treated infants ( a,c ) Quantified virus in blood and ( b,d ) peripheral blood cells in both NmAb dosing groups of male and female infant rhesus macaques ( n = 10). Plasma viral loads were assessed by measurements of SIV viral RNA in blood using a quantitative reverse-transcription PCR (QRT-PCR) assay and in ( b ) PBMC by quantitative PCR (QPCR). CD8 + T cell depletion study timeline is shown in red. Data shown in gray indicate mean plasma virus (+/− SD) from eight historical controls from an earlier study 18 , 30 .
Figure Legend Snippet: SHIV SF162P3 -associated viremia is not established in plasma or PBMC of NmAb-treated infants ( a,c ) Quantified virus in blood and ( b,d ) peripheral blood cells in both NmAb dosing groups of male and female infant rhesus macaques ( n = 10). Plasma viral loads were assessed by measurements of SIV viral RNA in blood using a quantitative reverse-transcription PCR (QRT-PCR) assay and in ( b ) PBMC by quantitative PCR (QPCR). CD8 + T cell depletion study timeline is shown in red. Data shown in gray indicate mean plasma virus (+/− SD) from eight historical controls from an earlier study 18 , 30 .

Techniques Used: Polymerase Chain Reaction, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

6) Product Images from "Performance of plate-based cytokine flow cytometry with automated data analysis"

Article Title: Performance of plate-based cytokine flow cytometry with automated data analysis

Journal: BMC Immunology

doi: 10.1186/1471-2172-4-9

Effect of overnight
Figure Legend Snippet: Effect of overnight "resting" on cryopreserved PBMC. (A) Representative comparison of fresh PBMC, cryopreserved PBMC, and cryopreserved PBMC incubated overnight prior to stimulation (all from the same CMV + donor). Unstimulated samples (top row) and CMV pp65 peptide mix-stimulated samples (bottom row) show similar results, but the IFNγ fluorescence intensity (MFI) is greatest in the cells rested overnight prior to stimulation. (B) Results of triplicate samples stimulated with CMV pp65 peptide mix and analyzed for percentage of IFNγ + cells or IFNγ mean fluorescence intensity. Error bars represent SEM. All data were gated on CD3 + CD4 - cells. Similar results were obtained for TNFα + cells (not shown). Results are representative of two similar experiments.

Techniques Used: Incubation, Fluorescence

Representative examples of tube- and plate-based CFC results. (A) Whole blood from a CMV seropositive donor was stimulated (or not) with CMV pp65 peptide mix in 15 ml conical polypropylene tubes (top panels) or a deep-well 96-well polypropylene plate (bottom panels). (B) PBMC from another CMV seropositive donor were stimulated as above in 15 ml conical polypropylene tubes (top panels) or a 96-well round-bottom tissue culture plate (bottom panels). (C) Whole blood from an HIV-seropositve donor was stimulated (or not) with HIV p55 gag peptide mix in 15 ml conical polypropylene tubes (top panels) or a deep-well 24-well round-bottom polypropylene plate (bottom panels). Backgrounds and response to peptide mix were essentially equivalent in tubes and plates in each case. All data are gated on CD3 + CD8 + cells.
Figure Legend Snippet: Representative examples of tube- and plate-based CFC results. (A) Whole blood from a CMV seropositive donor was stimulated (or not) with CMV pp65 peptide mix in 15 ml conical polypropylene tubes (top panels) or a deep-well 96-well polypropylene plate (bottom panels). (B) PBMC from another CMV seropositive donor were stimulated as above in 15 ml conical polypropylene tubes (top panels) or a 96-well round-bottom tissue culture plate (bottom panels). (C) Whole blood from an HIV-seropositve donor was stimulated (or not) with HIV p55 gag peptide mix in 15 ml conical polypropylene tubes (top panels) or a deep-well 24-well round-bottom polypropylene plate (bottom panels). Backgrounds and response to peptide mix were essentially equivalent in tubes and plates in each case. All data are gated on CD3 + CD8 + cells.

Techniques Used:

7) Product Images from "HDAC inhibitors decrease TLR7/9-mediated human plasmacytoid dendritic cell activation by interfering with IRF-7 and NF-κB signaling"

Article Title: HDAC inhibitors decrease TLR7/9-mediated human plasmacytoid dendritic cell activation by interfering with IRF-7 and NF-κB signaling

Journal: bioRxiv

doi: 10.1101/2020.05.09.085456

TSA, SAHA, and MS-275 decrease TLR9-induced activation of IRF-7 and NF-κB p65 transcription factors. PBMC were incubated with TSA (100 ng/ml) or DMSO (1:1000) for 1 hour, stimulated with HSV-1 (MOI of 1) or IAV (MOI of 2) for 3 hours. Samples were processed using BD Phosflow protocol. PBMC were gated based on SSC vs. FSC-A (Area) and pDC were identified by gating on +HLA-DRhigh/+CD123high events on a HLA-DR APC vs. CD123 PE intensity dot plot, then percent phosphorylated IRF-7+ pDC were gated based on mock sample for HSV-1 or IAV from which percentages were reported. Representative analysis for TSA, SAHA, and MS-275-mediated effect on IAV-mediated IRF-7 and NF-kB p65 phosphorylation in pDC. Red lines represent the position where the gate was placed from which percentages were derived. (A) . Pooled data with HSV-1 stimulation (top panels) and IAV (bottom panels) showing the effect of TSA (100 ng/ml) (B) , MS-275 (5 μM) (C) , and SAHA (500 nM) (D) , or DMSO (1:1000) on the phosphorylation of IRF-7 and NF-kB p65. Data represent the percentage of pIRF-7+ and pNF-κB p65+ pDC with gating based on mock sample. (N = 4-5, 4-5 independent experiments with different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; n.s. = not significant).
Figure Legend Snippet: TSA, SAHA, and MS-275 decrease TLR9-induced activation of IRF-7 and NF-κB p65 transcription factors. PBMC were incubated with TSA (100 ng/ml) or DMSO (1:1000) for 1 hour, stimulated with HSV-1 (MOI of 1) or IAV (MOI of 2) for 3 hours. Samples were processed using BD Phosflow protocol. PBMC were gated based on SSC vs. FSC-A (Area) and pDC were identified by gating on +HLA-DRhigh/+CD123high events on a HLA-DR APC vs. CD123 PE intensity dot plot, then percent phosphorylated IRF-7+ pDC were gated based on mock sample for HSV-1 or IAV from which percentages were reported. Representative analysis for TSA, SAHA, and MS-275-mediated effect on IAV-mediated IRF-7 and NF-kB p65 phosphorylation in pDC. Red lines represent the position where the gate was placed from which percentages were derived. (A) . Pooled data with HSV-1 stimulation (top panels) and IAV (bottom panels) showing the effect of TSA (100 ng/ml) (B) , MS-275 (5 μM) (C) , and SAHA (500 nM) (D) , or DMSO (1:1000) on the phosphorylation of IRF-7 and NF-kB p65. Data represent the percentage of pIRF-7+ and pNF-κB p65+ pDC with gating based on mock sample. (N = 4-5, 4-5 independent experiments with different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; n.s. = not significant).

Techniques Used: Activation Assay, Incubation, Derivative Assay

TSA and MS-275 inhibit HSV-1 and IAV-induced total IFN-α production in human pDC. PBMC were isolated and pre-treated with TSA (100 ng/ml) or DMSO vehicle control (1:1000) (A) and MS-275 (5 μM) or DMSO vehicle control (1:530) (B) for 1 hour and then stimulated with HSV-1 (MOI of 1) and IAV (MOI of 2) for 18 hours. Supernatants were collected and tested for IFN-α production by an ELISA assay. (N=4, 4 independent experiments from different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; *p
Figure Legend Snippet: TSA and MS-275 inhibit HSV-1 and IAV-induced total IFN-α production in human pDC. PBMC were isolated and pre-treated with TSA (100 ng/ml) or DMSO vehicle control (1:1000) (A) and MS-275 (5 μM) or DMSO vehicle control (1:530) (B) for 1 hour and then stimulated with HSV-1 (MOI of 1) and IAV (MOI of 2) for 18 hours. Supernatants were collected and tested for IFN-α production by an ELISA assay. (N=4, 4 independent experiments from different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; *p

Techniques Used: Isolation, Enzyme-linked Immunosorbent Assay

TSA and MS-275 inhibit the upregulation of pDC maturation markers upon TLR7/9 stimulation, and increase the shedding of the activation marker CD62L. PBMC were incubated with TSA (100 ng/ml), MS-275 (5 μM), or DMSO (1:530) for 1 hour and then stimulated with HSV-1 (MOI of 1), IAV (MOI of 2), or CpG-B (5 μg/ml) for 8 hours. Representative data derived from events first gated on PBMC, then pDC were identified by a BDCA-2 + /CD123 high gate. From this gate, histograms were derived and gates applied based on the unstimulated control in DMSO (Mock) (A) . Surface expression of CD62L (B, top) , CD40 (B, bottom) , CD86 (C, top), CD82 (C, bottom) were measured by flow cytometry and reported as percentage. (N = 4, 4 independent experiments with different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA and Bonferroni’s post test; *p
Figure Legend Snippet: TSA and MS-275 inhibit the upregulation of pDC maturation markers upon TLR7/9 stimulation, and increase the shedding of the activation marker CD62L. PBMC were incubated with TSA (100 ng/ml), MS-275 (5 μM), or DMSO (1:530) for 1 hour and then stimulated with HSV-1 (MOI of 1), IAV (MOI of 2), or CpG-B (5 μg/ml) for 8 hours. Representative data derived from events first gated on PBMC, then pDC were identified by a BDCA-2 + /CD123 high gate. From this gate, histograms were derived and gates applied based on the unstimulated control in DMSO (Mock) (A) . Surface expression of CD62L (B, top) , CD40 (B, bottom) , CD86 (C, top), CD82 (C, bottom) were measured by flow cytometry and reported as percentage. (N = 4, 4 independent experiments with different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA and Bonferroni’s post test; *p

Techniques Used: Activation Assay, Marker, Incubation, Derivative Assay, Expressing, Flow Cytometry

TSA and MS-275, but not SAHA, inhibits IAV and HSV-1-mediated IFN-α protein upregulation without decreasing IRF-7 upregulation. PBMC were pre-treated with TSA (100 ng/ml), SAHA (500 nM) and DMSO vehicle control (1:1000) or MS-275 (5 μM) and DMSO (1:530), for 1 hour and then stimulated with HSV-1 (MOI of 1) or IAV (MOI of 2) for 6 hours. IRF-7 (black bars) and intracellular IFN-α (gray bars) protein expression was measured concurrently by intracellular flow cytometry. Representative data showing the effect, on IAV-induced IFN (A, top) and IRF-7 (A, bottom) upregulation in pDC, by TSA, MS-275, and SAHA (A). Cumulative experiments showing the effect on HSV-1 (top) and IAV (bottom)-induced upregulation of IFN-α and IRF-7, by TSA (100 ng/ml) (B) and MS-275 (5 μM) (C) and SAHA (500 nM) (D). (N = 5; 5 independent experiments with different donors for IAV. N = 3; 3 independent experiments with different donors for HSV-1. Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; (* = p
Figure Legend Snippet: TSA and MS-275, but not SAHA, inhibits IAV and HSV-1-mediated IFN-α protein upregulation without decreasing IRF-7 upregulation. PBMC were pre-treated with TSA (100 ng/ml), SAHA (500 nM) and DMSO vehicle control (1:1000) or MS-275 (5 μM) and DMSO (1:530), for 1 hour and then stimulated with HSV-1 (MOI of 1) or IAV (MOI of 2) for 6 hours. IRF-7 (black bars) and intracellular IFN-α (gray bars) protein expression was measured concurrently by intracellular flow cytometry. Representative data showing the effect, on IAV-induced IFN (A, top) and IRF-7 (A, bottom) upregulation in pDC, by TSA, MS-275, and SAHA (A). Cumulative experiments showing the effect on HSV-1 (top) and IAV (bottom)-induced upregulation of IFN-α and IRF-7, by TSA (100 ng/ml) (B) and MS-275 (5 μM) (C) and SAHA (500 nM) (D). (N = 5; 5 independent experiments with different donors for IAV. N = 3; 3 independent experiments with different donors for HSV-1. Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; (* = p

Techniques Used: Expressing, Flow Cytometry

TSA and MS-275 inhibit HSV-1 and IAV-induced IFN-α and TNF-α production in human pDC in a dose-dependent manner. PBMC were incubated with TSA and MS-275 at decreasing concentrations for 1 hour and then stimulated with HSV-1 or IAV for 6 hours. Cells were then processed for intracellular flow cytometry to detect IFN-α and TNF-α production in pDC. Representative histograms showing the differential inhibition of intracellular HSV-1 and IAV-induced IFN-α and TNF-α production in pDC by TSA (100 ng/ml = 330 nM) or DMSO vehicle control (1:1000) (A), and MS-275 (5 μM) or DMSO vehicle control (1:530) (C). Pooled data of dose curve experiments showing a dose-dependent inhibitory effect on HSV-1- (MOI of 1; squares) or IAV (MOI of 2; triangles) -induced IFN-α and TNF-α by TSA (0-100 ng/ml) (C) and MS-275 (0-5 μM) (D) when compared to their respective vehicle controls. (TSA, N=3; MS-275, N=4; 3-4 independent experiments from different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; *p
Figure Legend Snippet: TSA and MS-275 inhibit HSV-1 and IAV-induced IFN-α and TNF-α production in human pDC in a dose-dependent manner. PBMC were incubated with TSA and MS-275 at decreasing concentrations for 1 hour and then stimulated with HSV-1 or IAV for 6 hours. Cells were then processed for intracellular flow cytometry to detect IFN-α and TNF-α production in pDC. Representative histograms showing the differential inhibition of intracellular HSV-1 and IAV-induced IFN-α and TNF-α production in pDC by TSA (100 ng/ml = 330 nM) or DMSO vehicle control (1:1000) (A), and MS-275 (5 μM) or DMSO vehicle control (1:530) (C). Pooled data of dose curve experiments showing a dose-dependent inhibitory effect on HSV-1- (MOI of 1; squares) or IAV (MOI of 2; triangles) -induced IFN-α and TNF-α by TSA (0-100 ng/ml) (C) and MS-275 (0-5 μM) (D) when compared to their respective vehicle controls. (TSA, N=3; MS-275, N=4; 3-4 independent experiments from different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; *p

Techniques Used: Incubation, Flow Cytometry, Inhibition

8) Product Images from "Generation of CD8+ T-Cell Responses by a Recombinant Nonpathogenic Mycobacterium smegmatis Vaccine Vector Expressing Human Immunodeficiency Virus Type 1 Env"

Article Title: Generation of CD8+ T-Cell Responses by a Recombinant Nonpathogenic Mycobacterium smegmatis Vaccine Vector Expressing Human Immunodeficiency Virus Type 1 Env

Journal: Journal of Virology

doi: 10.1128/JVI.80.4.1645-1652.2006

Recombinant M. smegmatis -elicited HIV-1-specific CD8 + T-cell responses in mice preimmunized with BCG. Mice were immunized with wild-type BCG (Pasteur) or PBS and 6 months later with 10 8 CFU r M. smegmatis expressing gp120 (integrative) (indicated as BCG + rSmeg-gp120 and PBS + rSmeg-gp120, respectively). BCG-preimmunized mice that were subsequently inoculated with rSmeg control were used as a negative control (indicated as BCG + rSmeg control). Tetramer analysis was performed on the PBMC of mice 1 week after inoculation with the r M. smegmatis constructs. The mean (± SEM) percent of HIV-1 HXBc2 gp120 P18-tetramer-positive CD8 + T cells is shown for each group ( n = 4 to 5 mice per group).
Figure Legend Snippet: Recombinant M. smegmatis -elicited HIV-1-specific CD8 + T-cell responses in mice preimmunized with BCG. Mice were immunized with wild-type BCG (Pasteur) or PBS and 6 months later with 10 8 CFU r M. smegmatis expressing gp120 (integrative) (indicated as BCG + rSmeg-gp120 and PBS + rSmeg-gp120, respectively). BCG-preimmunized mice that were subsequently inoculated with rSmeg control were used as a negative control (indicated as BCG + rSmeg control). Tetramer analysis was performed on the PBMC of mice 1 week after inoculation with the r M. smegmatis constructs. The mean (± SEM) percent of HIV-1 HXBc2 gp120 P18-tetramer-positive CD8 + T cells is shown for each group ( n = 4 to 5 mice per group).

Techniques Used: Recombinant, Mouse Assay, Expressing, Negative Control, Construct

Phenotype of HIV-1-specific CD8 T cells elicited by immunization with r M. smegmatis . Mice were immunized with 10 8 CFU r M. smegmatis expressing gp120 (integrative). (A) Flow cytometric analysis of week 1 PBMC and splenocytes from immunized mice revealed expression of CD44 on the surface of all r M. smegmatis -elicited tetramer-positive cells. CD62L and CD127 were expressed on a subset of the tetramer-positive cells. (B) The proportions of effector (P18-tetramer positive, CD127 − , and CD62L lo ), effector memory (P18-tetramer positive, CD127 + , and CD62L lo ), and central memory (P18-tetramer positive, CD127 + , and CD62L hi ) cells in the blood and spleen of mice immunized with r M. smegmatis are shown. Effector, effector memory, and central memory cells are denoted E, EM, and CM, respectively. The mean (± SEM) percent E, EM, or CM for each group of mice ( n = 4 per group) is shown. (C) Peripheral blood HIV-1-specific CD8 + T cells from mice 1 year after immunization with r M. smegmatis expressing gp120 were predominantly central memory cells. PBMC were pooled from 4 mice that were inoculated twice (10 weeks apart) with 10 8 CFU bacilli.
Figure Legend Snippet: Phenotype of HIV-1-specific CD8 T cells elicited by immunization with r M. smegmatis . Mice were immunized with 10 8 CFU r M. smegmatis expressing gp120 (integrative). (A) Flow cytometric analysis of week 1 PBMC and splenocytes from immunized mice revealed expression of CD44 on the surface of all r M. smegmatis -elicited tetramer-positive cells. CD62L and CD127 were expressed on a subset of the tetramer-positive cells. (B) The proportions of effector (P18-tetramer positive, CD127 − , and CD62L lo ), effector memory (P18-tetramer positive, CD127 + , and CD62L lo ), and central memory (P18-tetramer positive, CD127 + , and CD62L hi ) cells in the blood and spleen of mice immunized with r M. smegmatis are shown. Effector, effector memory, and central memory cells are denoted E, EM, and CM, respectively. The mean (± SEM) percent E, EM, or CM for each group of mice ( n = 4 per group) is shown. (C) Peripheral blood HIV-1-specific CD8 + T cells from mice 1 year after immunization with r M. smegmatis expressing gp120 were predominantly central memory cells. PBMC were pooled from 4 mice that were inoculated twice (10 weeks apart) with 10 8 CFU bacilli.

Techniques Used: Mouse Assay, Expressing, Flow Cytometry

Recombinant M. smegmatis elicited HIV-1-specific CD8 + T-cell responses in mice. BALB/c mice were inoculated via the intraperitoneal route with approximately 10 6 CFU or 10 8 CFU gp120-expressing recombinant M. smegmatis (rSmeg-gp120) organisms transformed with either the integrative pJH223- gp120 plasmid (A) or multicopy pJH222- gp120 (B). As a negative control, mice were inoculated with the same dose of mycobacteria transformed with the control pJH222- and pJH223- msp1 plasmids (rSmeg control). (C) Mice were inoculated twice (10 weeks apart) with the same dose of either the rSmeg-gp120 (integrative) construct or the rSmeg control. The mean (± SEM) percent HIV-1 HXBc2 gp120 P18 tetramer-positive CD8 T cells from PBMC collected at the indicated time points is shown for each group of mice ( n = 4 per group).
Figure Legend Snippet: Recombinant M. smegmatis elicited HIV-1-specific CD8 + T-cell responses in mice. BALB/c mice were inoculated via the intraperitoneal route with approximately 10 6 CFU or 10 8 CFU gp120-expressing recombinant M. smegmatis (rSmeg-gp120) organisms transformed with either the integrative pJH223- gp120 plasmid (A) or multicopy pJH222- gp120 (B). As a negative control, mice were inoculated with the same dose of mycobacteria transformed with the control pJH222- and pJH223- msp1 plasmids (rSmeg control). (C) Mice were inoculated twice (10 weeks apart) with the same dose of either the rSmeg-gp120 (integrative) construct or the rSmeg control. The mean (± SEM) percent HIV-1 HXBc2 gp120 P18 tetramer-positive CD8 T cells from PBMC collected at the indicated time points is shown for each group of mice ( n = 4 per group).

Techniques Used: Recombinant, Mouse Assay, Expressing, Transformation Assay, Plasmid Preparation, Negative Control, Construct

9) Product Images from "Accessory cell dependent NK cell mediated PBMC IFN-γ production is defective in HIV infection"

Article Title: Accessory cell dependent NK cell mediated PBMC IFN-γ production is defective in HIV infection

Journal: Clinical immunology (Orlando, Fla.)

doi: 10.1016/j.clim.2008.12.012

Poly (I:C) induced IFN-γ production is dependent on MDC, and in part dependent on IL-12. Panel A: CD3 depleted PBMC from healthy control subjects ( n =5) were stimulated with Poly(I:C) (50 µg/ml, 20 h 37 °C) in the presence or absence
Figure Legend Snippet: Poly (I:C) induced IFN-γ production is dependent on MDC, and in part dependent on IL-12. Panel A: CD3 depleted PBMC from healthy control subjects ( n =5) were stimulated with Poly(I:C) (50 µg/ml, 20 h 37 °C) in the presence or absence

Techniques Used:

Poly (I:C) induced CD3 depleted PBMC IFN-γ is impaired in HIV and HCV-HIV infections, while IL-12 induced activity is intact
Figure Legend Snippet: Poly (I:C) induced CD3 depleted PBMC IFN-γ is impaired in HIV and HCV-HIV infections, while IL-12 induced activity is intact

Techniques Used: Activity Assay

Poly (I:C) induced CD3 depleted PBMC IFN-γ is impaired in HIV infection, and this activity is associated with IL-12. Panel A: PBMC were cultured in the absence (media) or presence of poly (I:C) (50 µg/ml) or IL-12 (1 ng/ml) for 20 h. Cells
Figure Legend Snippet: Poly (I:C) induced CD3 depleted PBMC IFN-γ is impaired in HIV infection, and this activity is associated with IL-12. Panel A: PBMC were cultured in the absence (media) or presence of poly (I:C) (50 µg/ml) or IL-12 (1 ng/ml) for 20 h. Cells

Techniques Used: Infection, Activity Assay, Cell Culture

10) Product Images from "Porcine CD3+NKp46+ Lymphocytes Have NK-Cell Characteristics and Are Present in Increased Frequencies in the Lungs of Influenza-Infected Animals"

Article Title: Porcine CD3+NKp46+ Lymphocytes Have NK-Cell Characteristics and Are Present in Increased Frequencies in the Lungs of Influenza-Infected Animals

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2016.00263

Proliferative capacity of CD3 + NKp46 + lymphocytes . PBMC were stained with Violet Cell Trace Dye to analyze proliferating cells following stimulation with either ConA or rpIL-2 and rpIL-15 for 4 days. Cells cultured in medium alone served as negative control. Proliferation of CD3 + NKp46 + lymphocytes (red) was compared with CD3 + NKp46 − T cells (gray) and total CD3 − NK cells (green). For NK cells, an additional pre-gating on CD3 − CD16 + was performed. (A) Histograms show proliferation of the different lymphocyte subsets following respective stimulation for one representative animal. Percentages of proliferating cells within respective subsets are indicated. (B) Frequencies of proliferating cells within the three analyzed lymphocyte subsets in blood of six animals are shown. Mean values are represented by colored bars. Significant differences between the CD3 + NKp46 + cells and T as well as NK cells are indicated (*** p ≤ 0.001).
Figure Legend Snippet: Proliferative capacity of CD3 + NKp46 + lymphocytes . PBMC were stained with Violet Cell Trace Dye to analyze proliferating cells following stimulation with either ConA or rpIL-2 and rpIL-15 for 4 days. Cells cultured in medium alone served as negative control. Proliferation of CD3 + NKp46 + lymphocytes (red) was compared with CD3 + NKp46 − T cells (gray) and total CD3 − NK cells (green). For NK cells, an additional pre-gating on CD3 − CD16 + was performed. (A) Histograms show proliferation of the different lymphocyte subsets following respective stimulation for one representative animal. Percentages of proliferating cells within respective subsets are indicated. (B) Frequencies of proliferating cells within the three analyzed lymphocyte subsets in blood of six animals are shown. Mean values are represented by colored bars. Significant differences between the CD3 + NKp46 + cells and T as well as NK cells are indicated (*** p ≤ 0.001).

Techniques Used: Staining, Cell Culture, Negative Control

CD3 + NKp46 + lymphocytes in influenza A-infected piglets . CD3 + NKp46 + lymphocytes from blood and lung of influenza A-infected piglets and healthy control animals were analyzed by flow cytometry on days 1–3 after infection. (A) Absolute numbers of CD3 + NKp46 + cells in PBMC of controls (left, n = 9) and infected animals (right, n = 12) are shown in the course of influenza infection. (B) Box-plots show absolute numbers of CD3 + NKp46 + cells in PBMC on individual days post-infection. (C) Frequencies of CD3 + NKp46 + cells among lung lymphocytes in control ( n = 6) and infected animals ( n = 12) in the course of influenza infection. (D) Box-plots show median fluorescence intensity of Ki-67 in CD3 + NKp46 + lung cells. Significant differences between infected and control animals as well as distinct study days are indicated (* p ≤ 0.05, ** p ≤ 0.01).
Figure Legend Snippet: CD3 + NKp46 + lymphocytes in influenza A-infected piglets . CD3 + NKp46 + lymphocytes from blood and lung of influenza A-infected piglets and healthy control animals were analyzed by flow cytometry on days 1–3 after infection. (A) Absolute numbers of CD3 + NKp46 + cells in PBMC of controls (left, n = 9) and infected animals (right, n = 12) are shown in the course of influenza infection. (B) Box-plots show absolute numbers of CD3 + NKp46 + cells in PBMC on individual days post-infection. (C) Frequencies of CD3 + NKp46 + cells among lung lymphocytes in control ( n = 6) and infected animals ( n = 12) in the course of influenza infection. (D) Box-plots show median fluorescence intensity of Ki-67 in CD3 + NKp46 + lung cells. Significant differences between infected and control animals as well as distinct study days are indicated (* p ≤ 0.05, ** p ≤ 0.01).

Techniques Used: Infection, Flow Cytometry, Cytometry, Fluorescence

Activation of CD3 + NKp46 + lymphocytes by receptor cross-linking . Receptor-mediated degranulation, IFN-γ production, and proliferation was assessed by multicolor FCM in response to cross-linking of NKp46 or CD3 by plate-bound mAbs. Isotype-matched irrelevant antibodies served as control. Activation of CD3 + NKp46 + lymphocytes (red) derived from blood of four different individuals was analyzed and compared with either total CD3 − NK cells (green) or CD3 + NKp46 − T cells (gray). Gating for FCM analyses was performed as displayed in the previous figures. (A) PBMC were pre-activated with rpIL-2 and rpIL-15 for 24 h and receptor-mediated degranulation was assessed by CD107a expression on the cell surface after 4 h incubation with plate-bound mAbs. (B) PBMC were pre-activated with rpIL-2 and rpIL-18 for 24 h and intracellular IFN-γ production was measured after receptor cross-linking for 4 h. (C) Total PBMC were stained with Violet Cell Trace Dye and cultured for 4 days in the presence of plate-bound mAbs. (A–C) Dot-plots and histograms show results of CD3 + NKp46 + lymphocytes for one representative animal. CD3 + NKp46 + cells are shown in red, total lymphocytes are shown in light gray as background. Frequencies of CD107a + and IFN-γ + as well as proliferating cells within lymphocyte subsets are shown for analyses of four animals in the graphs. Mean values are represented by colored bars. Significant differences between stimulated and non-stimulated CD3 + NKp46 + cells, T and NK cells are indicated (* p ≤ 0.05, ** p ≤ 0.01).
Figure Legend Snippet: Activation of CD3 + NKp46 + lymphocytes by receptor cross-linking . Receptor-mediated degranulation, IFN-γ production, and proliferation was assessed by multicolor FCM in response to cross-linking of NKp46 or CD3 by plate-bound mAbs. Isotype-matched irrelevant antibodies served as control. Activation of CD3 + NKp46 + lymphocytes (red) derived from blood of four different individuals was analyzed and compared with either total CD3 − NK cells (green) or CD3 + NKp46 − T cells (gray). Gating for FCM analyses was performed as displayed in the previous figures. (A) PBMC were pre-activated with rpIL-2 and rpIL-15 for 24 h and receptor-mediated degranulation was assessed by CD107a expression on the cell surface after 4 h incubation with plate-bound mAbs. (B) PBMC were pre-activated with rpIL-2 and rpIL-18 for 24 h and intracellular IFN-γ production was measured after receptor cross-linking for 4 h. (C) Total PBMC were stained with Violet Cell Trace Dye and cultured for 4 days in the presence of plate-bound mAbs. (A–C) Dot-plots and histograms show results of CD3 + NKp46 + lymphocytes for one representative animal. CD3 + NKp46 + cells are shown in red, total lymphocytes are shown in light gray as background. Frequencies of CD107a + and IFN-γ + as well as proliferating cells within lymphocyte subsets are shown for analyses of four animals in the graphs. Mean values are represented by colored bars. Significant differences between stimulated and non-stimulated CD3 + NKp46 + cells, T and NK cells are indicated (* p ≤ 0.05, ** p ≤ 0.01).

Techniques Used: Activation Assay, Derivative Assay, Expressing, Incubation, Staining, Cell Culture

Expression of NK-associated receptor mRNAs in CD3 + NKp46 + lymphocytes . (A) PBMC were FACS sorted into CD3 + NKp46 − T cells (gray), CD3 + NKp46 + cells (red), and total CD3 − NK cells (green). (B) Sorted lymphocyte subsets were analyzed for their mRNA expression levels of NKp30, NKp44, and NKG2D by quantitative RT-PCR. The 2 −ΔΔCq values for the target genes of each individual animal ( n = 4) are shown as fold differences relative to the mean of CD3 + NKp46 + cells from all animals, which was set to a value of 1. Colored lines represent mean values of the respective lymphocyte subsets. Significant differences between the CD3 + NKp46 + cells and T as well as NK cells are indicated (* p ≤ 0.05, *** p ≤ 0.001).
Figure Legend Snippet: Expression of NK-associated receptor mRNAs in CD3 + NKp46 + lymphocytes . (A) PBMC were FACS sorted into CD3 + NKp46 − T cells (gray), CD3 + NKp46 + cells (red), and total CD3 − NK cells (green). (B) Sorted lymphocyte subsets were analyzed for their mRNA expression levels of NKp30, NKp44, and NKG2D by quantitative RT-PCR. The 2 −ΔΔCq values for the target genes of each individual animal ( n = 4) are shown as fold differences relative to the mean of CD3 + NKp46 + cells from all animals, which was set to a value of 1. Colored lines represent mean values of the respective lymphocyte subsets. Significant differences between the CD3 + NKp46 + cells and T as well as NK cells are indicated (* p ≤ 0.05, *** p ≤ 0.001).

Techniques Used: Expressing, FACS, Quantitative RT-PCR

IFN-γ production of CD3 + NKp46 + lymphocytes . PBMC were stimulated either with PMA/Ionomycin for 4 h or with a combination of rpIL-2 + rpIL-12 + rpIL-18 for 24 h. IL-18 was used at two different concentrations (low: 5 ng/ml, high: 25 ng/ml). Cells cultured in medium alone served as negative control. Following stimulation, intracellular IFN-γ expression of CD3 + NKp46 + lymphocytes (red) was analyzed and compared with CD3 + NKp46 − T cells (gray) and total CD3 − NK cells (green). (A) IFN-γ expression of the respective lymphocyte subsets is shown in the dot-plots on the right in combination with CD8β for one representative animal. Analyzed cell subsets are highlighted in color, total lymphocyte population is shown in light gray in the background. Percentages of IFN-γ + cells within respective subsets are indicated. (B) Frequencies of IFN-γ + cells (upper row) as well as median fluorescence intensity of IFN-γ + cells (bottom row) within the three analyzed lymphocyte subsets in blood of six animals are shown. Mean values are represented by colored bars. Significant differences between the CD3 + NKp46 + cells and T as well as NK cells are indicated (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).
Figure Legend Snippet: IFN-γ production of CD3 + NKp46 + lymphocytes . PBMC were stimulated either with PMA/Ionomycin for 4 h or with a combination of rpIL-2 + rpIL-12 + rpIL-18 for 24 h. IL-18 was used at two different concentrations (low: 5 ng/ml, high: 25 ng/ml). Cells cultured in medium alone served as negative control. Following stimulation, intracellular IFN-γ expression of CD3 + NKp46 + lymphocytes (red) was analyzed and compared with CD3 + NKp46 − T cells (gray) and total CD3 − NK cells (green). (A) IFN-γ expression of the respective lymphocyte subsets is shown in the dot-plots on the right in combination with CD8β for one representative animal. Analyzed cell subsets are highlighted in color, total lymphocyte population is shown in light gray in the background. Percentages of IFN-γ + cells within respective subsets are indicated. (B) Frequencies of IFN-γ + cells (upper row) as well as median fluorescence intensity of IFN-γ + cells (bottom row) within the three analyzed lymphocyte subsets in blood of six animals are shown. Mean values are represented by colored bars. Significant differences between the CD3 + NKp46 + cells and T as well as NK cells are indicated (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).

Techniques Used: Cell Culture, Negative Control, Expressing, Fluorescence

Cytolytic properties of CD3 + NKp46 + lymphocytes . (A) Lymphocytes derived from blood and lung were analyzed for perforin expression. Histograms show the perforin expression levels of CD3 + NKp46 + cells (red) compared with CD3 + NKp46 − T cells (gray) and total CD3 − NK cells (green) for one representative animal. Results for CD3 − CD8α − cells, mainly consisting of B cells, are shown in light gray and serve as negative reference. (B) Median fluorescence intensities of perforin within respective lymphocyte subsets are shown for PBMC and lung for six animals. Mean values are represented by colored bars. Significant differences between the CD3 + NKp46 + cells and T as well as NK cells are indicated (** p ≤ 0.01, *** p ≤ 0.001). (C) FACS-sorted CD3 + NKp46 − T cells (gray), CD3 + NKp46 + cells (red), and total CD3 − NK cells (green) from blood were stimulated with rpIL-2 and rpIL-15 for 36 h and subsequently used in a 4h-cytotoxic assay with K562 as target cell line. Respective lymphocyte subsets were tested at five different E:T ratios: 20:1, 10:1, 5:1, 2.5:1, and 1.25:1. Results obtained from analyses of four animals are shown. Colored lines represent mean values of the respective lymphocyte subsets.
Figure Legend Snippet: Cytolytic properties of CD3 + NKp46 + lymphocytes . (A) Lymphocytes derived from blood and lung were analyzed for perforin expression. Histograms show the perforin expression levels of CD3 + NKp46 + cells (red) compared with CD3 + NKp46 − T cells (gray) and total CD3 − NK cells (green) for one representative animal. Results for CD3 − CD8α − cells, mainly consisting of B cells, are shown in light gray and serve as negative reference. (B) Median fluorescence intensities of perforin within respective lymphocyte subsets are shown for PBMC and lung for six animals. Mean values are represented by colored bars. Significant differences between the CD3 + NKp46 + cells and T as well as NK cells are indicated (** p ≤ 0.01, *** p ≤ 0.001). (C) FACS-sorted CD3 + NKp46 − T cells (gray), CD3 + NKp46 + cells (red), and total CD3 − NK cells (green) from blood were stimulated with rpIL-2 and rpIL-15 for 36 h and subsequently used in a 4h-cytotoxic assay with K562 as target cell line. Respective lymphocyte subsets were tested at five different E:T ratios: 20:1, 10:1, 5:1, 2.5:1, and 1.25:1. Results obtained from analyses of four animals are shown. Colored lines represent mean values of the respective lymphocyte subsets.

Techniques Used: Derivative Assay, Expressing, Fluorescence, FACS

Porcine CD3 + NKp46 + lymphocytes in lymphatic and non-lymphatic organs . (A) Lymphocytes were gated according to their light scatter properties. Potential doublet cells were excluded by consecutive FSC-H/FSC-W and SSC-H/SSC-W gates, followed by gating of Near-IR − cells to exclude dead cells. Live lymphocytes were separated into CD3 − non-T cells to analyze CD8α/NKp46-defined NK-cell subsets (CD8α + NKp46 − , CD8α + NKp46 + and CD8α dim/− NKp46 high , green gates on the left) and CD3 + T cells to analyze CD3 + NKp46 + lymphocytes (red gate on the right). The gating strategy is shown as a representative example for PBMC and was performed correspondingly for all organs analyzed. (B) The three CD8α/NKp46-defined NK-cell subsets as well as the CD3 + NKp46 + lymphocytes (shown in red) in blood of 50 individuals were analyzed for their NKp46 (top) and CD8α (bottom) expression levels. Box-plots show the median fluorescence intensities of both markers within respective subsets. Significant differences between the CD3 + NKp46 + cells and NK-cell subsets are indicated (*** p ≤ 0.001). (C) Box-plots show the frequencies of the four CD3/CD8α/NKp46-defined lymphocyte subsets in blood ( n = 50). CD3 + NKp46 + cells are indicated in red. (D) CD3 + NKp46 + cells (red gates) were analyzed in lymphocytes isolated from blood ( n = 50), spleen ( n = 40), mediastinal lymph node (medLN, n = 20), liver ( n = 10), and lung ( n = 10). Representative examples of one animal are shown in the contour-plots. Box-plots show frequencies of CD3 + NKp46 + lymphocytes within respective organs. All results were obtained from healthy 3- to 7-month-old pigs.
Figure Legend Snippet: Porcine CD3 + NKp46 + lymphocytes in lymphatic and non-lymphatic organs . (A) Lymphocytes were gated according to their light scatter properties. Potential doublet cells were excluded by consecutive FSC-H/FSC-W and SSC-H/SSC-W gates, followed by gating of Near-IR − cells to exclude dead cells. Live lymphocytes were separated into CD3 − non-T cells to analyze CD8α/NKp46-defined NK-cell subsets (CD8α + NKp46 − , CD8α + NKp46 + and CD8α dim/− NKp46 high , green gates on the left) and CD3 + T cells to analyze CD3 + NKp46 + lymphocytes (red gate on the right). The gating strategy is shown as a representative example for PBMC and was performed correspondingly for all organs analyzed. (B) The three CD8α/NKp46-defined NK-cell subsets as well as the CD3 + NKp46 + lymphocytes (shown in red) in blood of 50 individuals were analyzed for their NKp46 (top) and CD8α (bottom) expression levels. Box-plots show the median fluorescence intensities of both markers within respective subsets. Significant differences between the CD3 + NKp46 + cells and NK-cell subsets are indicated (*** p ≤ 0.001). (C) Box-plots show the frequencies of the four CD3/CD8α/NKp46-defined lymphocyte subsets in blood ( n = 50). CD3 + NKp46 + cells are indicated in red. (D) CD3 + NKp46 + cells (red gates) were analyzed in lymphocytes isolated from blood ( n = 50), spleen ( n = 40), mediastinal lymph node (medLN, n = 20), liver ( n = 10), and lung ( n = 10). Representative examples of one animal are shown in the contour-plots. Box-plots show frequencies of CD3 + NKp46 + lymphocytes within respective organs. All results were obtained from healthy 3- to 7-month-old pigs.

Techniques Used: Expressing, Fluorescence, Isolation

11) Product Images from "SSX2 regulates focal adhesion but does not drive the epithelial to mesenchymal transition in prostate cancer"

Article Title: SSX2 regulates focal adhesion but does not drive the epithelial to mesenchymal transition in prostate cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.9802

SSX2 is expressed in metastases and circulating tumor cells of prostate cancer patients cDNA libraries from 15 metastatic prostate cancer samples were evaluated for SSX gene expression using primers specific for each SSX family member. A. Representative agarose gel of SSX2 expression Key: S = SSX2, A = actin, L = DNA marker ladder. B. Summary of findings for all SSX family members in cDNA from metastatic tissues. C. SSX2 mRNA was detected in the blood of patients with recurrent prostate cancer by PCR using primers specific for SSX2. Key: D0 = non-castrate, non-metastatic; D0.5 = castrate-resistant, non-metastatic; D2 = castrate-sensitive, metastatic; D3 = castrate-resistant, metastatic. PBMC previously found positive for SSX2 expression were FACS sorted based on expression of cell surface markers. Quantification of SSX2 expression was performed in CD45 + or CD45 − populations D. and CD45 + or CD45 − /EpCAM + /CD63 + (CTC) populations E. * = P
Figure Legend Snippet: SSX2 is expressed in metastases and circulating tumor cells of prostate cancer patients cDNA libraries from 15 metastatic prostate cancer samples were evaluated for SSX gene expression using primers specific for each SSX family member. A. Representative agarose gel of SSX2 expression Key: S = SSX2, A = actin, L = DNA marker ladder. B. Summary of findings for all SSX family members in cDNA from metastatic tissues. C. SSX2 mRNA was detected in the blood of patients with recurrent prostate cancer by PCR using primers specific for SSX2. Key: D0 = non-castrate, non-metastatic; D0.5 = castrate-resistant, non-metastatic; D2 = castrate-sensitive, metastatic; D3 = castrate-resistant, metastatic. PBMC previously found positive for SSX2 expression were FACS sorted based on expression of cell surface markers. Quantification of SSX2 expression was performed in CD45 + or CD45 − populations D. and CD45 + or CD45 − /EpCAM + /CD63 + (CTC) populations E. * = P

Techniques Used: Expressing, Agarose Gel Electrophoresis, Marker, Polymerase Chain Reaction, FACS

12) Product Images from "Intrabronchial Infection of Rhesus Macaques with Simian Varicella Virus Results in a Robust Immune Response in the Lungs"

Article Title: Intrabronchial Infection of Rhesus Macaques with Simian Varicella Virus Results in a Robust Immune Response in the Lungs

Journal: Journal of Virology

doi: 10.1128/JVI.01814-14

The frequency of plasmacytoid DCs increases early during acute SVV infection in the lungs. The frequencies (means ± SEM) of dendritic cells (DCs; lin − CD14 − HLA-DR + ) and monocytes (lin − CD14 + HLA-DR − ) in BAL cells (A) and PBMC (B) were measured by flow cytometry (++, P
Figure Legend Snippet: The frequency of plasmacytoid DCs increases early during acute SVV infection in the lungs. The frequencies (means ± SEM) of dendritic cells (DCs; lin − CD14 − HLA-DR + ) and monocytes (lin − CD14 + HLA-DR − ) in BAL cells (A) and PBMC (B) were measured by flow cytometry (++, P

Techniques Used: Infection, Flow Cytometry, Cytometry

Acute SVV infection results in upregulation of granzyme B in CD4 and CD8 T cell subsets in the lungs. The frequencies (means ± SEM) of granzyme B-positive T cells within CM, TEM, and EM subsets of CD4 (A and B) and CD8 (C and D) T cells were assessed by flow cytometry in BAL cells (A and C) and PBMC (B and D) (++, P
Figure Legend Snippet: Acute SVV infection results in upregulation of granzyme B in CD4 and CD8 T cell subsets in the lungs. The frequencies (means ± SEM) of granzyme B-positive T cells within CM, TEM, and EM subsets of CD4 (A and B) and CD8 (C and D) T cells were assessed by flow cytometry in BAL cells (A and C) and PBMC (B and D) (++, P

Techniques Used: Infection, Transmission Electron Microscopy, Flow Cytometry, Cytometry

Acute SVV infection elicits B cell proliferation and antibody production in the lungs. The frequency (mean ± SEM) of marginal zone (MZ)-like (IgD + CD27 + ), class-switched memory (IgD − CD27 + ), and double-negative (DN; IgD − CD27 − ) B cells was measured in BAL (A) and PBMC (B) samples by flow cytometry. Proliferation (means ± SEM) of B cell subsets was measured by detection of Ki67 in BAL (C) and PBMC (D) by flow cytometry ( , P
Figure Legend Snippet: Acute SVV infection elicits B cell proliferation and antibody production in the lungs. The frequency (mean ± SEM) of marginal zone (MZ)-like (IgD + CD27 + ), class-switched memory (IgD − CD27 + ), and double-negative (DN; IgD − CD27 − ) B cells was measured in BAL (A) and PBMC (B) samples by flow cytometry. Proliferation (means ± SEM) of B cell subsets was measured by detection of Ki67 in BAL (C) and PBMC (D) by flow cytometry ( , P

Techniques Used: Infection, Flow Cytometry, Cytometry

Acute SVV infection induces robust proliferation of CD4 and CD8 T cells in the lungs. The frequencies (means ± SEM) of central memory (CM; CD95 + CD28 + CCR7 + ), transitional effector memory (TEM; CD95 + CD28 + CCR7 − ), and effector memory (EM; CD95 + CD28 − CCR7 − ) subsets in CD4 (A, B) and CD8 (C, D) T cells in BAL cells (A, C) and PBMC (B, D) were measured by flow cytometry. Proliferation (mean ± SEM) of T cell subsets was measured by the expression of Ki67 in CD4 (E, F) and CD8 (G, H) T cells in BAL cells (E, G) and PBMC (F, H) (+, P
Figure Legend Snippet: Acute SVV infection induces robust proliferation of CD4 and CD8 T cells in the lungs. The frequencies (means ± SEM) of central memory (CM; CD95 + CD28 + CCR7 + ), transitional effector memory (TEM; CD95 + CD28 + CCR7 − ), and effector memory (EM; CD95 + CD28 − CCR7 − ) subsets in CD4 (A, B) and CD8 (C, D) T cells in BAL cells (A, C) and PBMC (B, D) were measured by flow cytometry. Proliferation (mean ± SEM) of T cell subsets was measured by the expression of Ki67 in CD4 (E, F) and CD8 (G, H) T cells in BAL cells (E, G) and PBMC (F, H) (+, P

Techniques Used: Infection, Transmission Electron Microscopy, Flow Cytometry, Cytometry, Expressing

13) Product Images from "Mechanisms of Gastrointestinal CD4+ T-Cell Depletion during Acute and Early Human Immunodeficiency Virus Type 1 Infection ▿"

Article Title: Mechanisms of Gastrointestinal CD4+ T-Cell Depletion during Acute and Early Human Immunodeficiency Virus Type 1 Infection ▿

Journal: Journal of Virology

doi: 10.1128/JVI.01739-06

Gastrointestinal CD4 + T cells harbor a greater viral burden than PB CD4 + T cells during acute and early HIV-1 infection. PBMC and MMCs from subjects with acute and early HIV-1 infection were flow cytometrically sorted with > 99.5%
Figure Legend Snippet: Gastrointestinal CD4 + T cells harbor a greater viral burden than PB CD4 + T cells during acute and early HIV-1 infection. PBMC and MMCs from subjects with acute and early HIV-1 infection were flow cytometrically sorted with > 99.5%

Techniques Used: Infection, Flow Cytometry

Immunological phenotype of PBMC and MMCs during acute and early HIV-1 infection. (A) Representative flow cytometry plots comparing effector memory cells between an HIV-uninfected control (upper panels) and a subject with acute HIV-1 infection (lower panels).
Figure Legend Snippet: Immunological phenotype of PBMC and MMCs during acute and early HIV-1 infection. (A) Representative flow cytometry plots comparing effector memory cells between an HIV-uninfected control (upper panels) and a subject with acute HIV-1 infection (lower panels).

Techniques Used: Infection, Flow Cytometry, Cytometry

14) Product Images from "CD8+ Lymphocytes Suppress Human Immunodeficiency Virus 1 Replication by Secreting Type I Interferons"

Article Title: CD8+ Lymphocytes Suppress Human Immunodeficiency Virus 1 Replication by Secreting Type I Interferons

Journal: Journal of Interferon & Cytokine Research

doi: 10.1089/jir.2012.0067

Low-level IFN in primary CD8 + cells. (A) Dot plots, gated on lymphocytes, show the profiles of unstimulated peripheral blood mononuclear cells after cell-surface staining with anti-CD8 and intracellular staining with an isotype control antibody ( upper
Figure Legend Snippet: Low-level IFN in primary CD8 + cells. (A) Dot plots, gated on lymphocytes, show the profiles of unstimulated peripheral blood mononuclear cells after cell-surface staining with anti-CD8 and intracellular staining with an isotype control antibody ( upper

Techniques Used: Staining

15) Product Images from "HCMV triggers frequent and persistent UL40-specific unconventional HLA-E-restricted CD8 T-cell responses with potential autologous and allogeneic peptide recognition"

Article Title: HCMV triggers frequent and persistent UL40-specific unconventional HLA-E-restricted CD8 T-cell responses with potential autologous and allogeneic peptide recognition

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007041

Time course analysis of the HLA-E UL40 and HLA-A*02 pp65 CD8 T-cell anti-HCMV responses upon infection and patterns of activation markers. (A) Time course analysis of the HLA-E UL40 and HLA-A*02 pp65 CD8 T-cell responses according to the HCMV viremia. PBMCs prospectively collected from M0 and M13 (#109) post-transplantation were retrospectively processed for the concomitant detection and quantification of anti-HCMV HLA-E UL40 and HLA-A*02 pp65 CD8 T-cell responses upon infection. Three representative patterns of anti-HCMV CD8 T cell responses in 3 KTR (KTR#107, #108 and #109) are represented. (B) Analysis of T-cell activation. Expression of CD69 (left panel) and PD-1 (right panel) analysed on blood samples from KTR#107, #108 and #109. Facs histogram overlays represent the % of expression for the activation markers CD69 and PD-1 among CD3 + CD8α + TCRγδ - tetramers + cells, for HLA-E UL40 (in blue) and HLA-A*02 pp65 (in red) anti-HCMV CD8 T-cell responses at M6 post-transplantation. (C) Comparative analysis of CD69 (left panel) and PD-1 (right panel) expression on HLA-E UL40 (n = 4 hosts) and HLA-A*02 pp65 (n = 8 hosts) CD8 T cells investigated at M6 post-transplantation. P values were calculated using a Mann Whitney test.
Figure Legend Snippet: Time course analysis of the HLA-E UL40 and HLA-A*02 pp65 CD8 T-cell anti-HCMV responses upon infection and patterns of activation markers. (A) Time course analysis of the HLA-E UL40 and HLA-A*02 pp65 CD8 T-cell responses according to the HCMV viremia. PBMCs prospectively collected from M0 and M13 (#109) post-transplantation were retrospectively processed for the concomitant detection and quantification of anti-HCMV HLA-E UL40 and HLA-A*02 pp65 CD8 T-cell responses upon infection. Three representative patterns of anti-HCMV CD8 T cell responses in 3 KTR (KTR#107, #108 and #109) are represented. (B) Analysis of T-cell activation. Expression of CD69 (left panel) and PD-1 (right panel) analysed on blood samples from KTR#107, #108 and #109. Facs histogram overlays represent the % of expression for the activation markers CD69 and PD-1 among CD3 + CD8α + TCRγδ - tetramers + cells, for HLA-E UL40 (in blue) and HLA-A*02 pp65 (in red) anti-HCMV CD8 T-cell responses at M6 post-transplantation. (C) Comparative analysis of CD69 (left panel) and PD-1 (right panel) expression on HLA-E UL40 (n = 4 hosts) and HLA-A*02 pp65 (n = 8 hosts) CD8 T cells investigated at M6 post-transplantation. P values were calculated using a Mann Whitney test.

Techniques Used: Infection, Activation Assay, Transplantation Assay, Expressing, FACS, MANN-WHITNEY

Potential cross-recognition of autologous and allogeneic HLA-I signal peptides by HLA-E UL40 CD8 T cells. PBMCs were isolated from freshly or prospectively collected blood samples at M12 post-transplantation issued from healthy donors (HV, n = 25) or from kidney transplant recipients (KTR, n = 119), respectively. Ex vivo detection of HLA-E UL40 CD8 T and HLA-A*02 pp65 -specific CD8 T cells was performed using flow cytometry by selecting CD3 + CD8α + TCRγδ - tetramer + cells on PBMCs. Eight different HLA-E UL40 tetramers were used independently. (A) Percentage of circulating anti-HCMV CD8 T cells in blood detected using the various HLA-E UL40 (blue) and HLA-A*02 pp65 (in red) tetramers in HV and KTR. For each tetramer/peptide, the number of individuals with a given CD8 T-cell response is indicated. (B) Diversity and magnitude of the HLA-E UL40 CD8 T-cell responses in KTR and HV. HLA-E UL40 CD8 T-cell responses appear in blue and colour intensity is proportional to the percentage of HLA-E UL40 CD8 T cells. (C) Classification of the HLA-E UL40 CD8 T-cell responses in HCMV + hosts according to possible recognition of self (orange), donor-specific allogeneic (green) or both (violet) (n = 31, 23 KTR and 8 HV). Grey boxes show HLA-I signal peptides which are not derived from the recipient, nor from the donor. Asterisks indicate peptides with underestimated information due to a lack of HLA-C genotyping.
Figure Legend Snippet: Potential cross-recognition of autologous and allogeneic HLA-I signal peptides by HLA-E UL40 CD8 T cells. PBMCs were isolated from freshly or prospectively collected blood samples at M12 post-transplantation issued from healthy donors (HV, n = 25) or from kidney transplant recipients (KTR, n = 119), respectively. Ex vivo detection of HLA-E UL40 CD8 T and HLA-A*02 pp65 -specific CD8 T cells was performed using flow cytometry by selecting CD3 + CD8α + TCRγδ - tetramer + cells on PBMCs. Eight different HLA-E UL40 tetramers were used independently. (A) Percentage of circulating anti-HCMV CD8 T cells in blood detected using the various HLA-E UL40 (blue) and HLA-A*02 pp65 (in red) tetramers in HV and KTR. For each tetramer/peptide, the number of individuals with a given CD8 T-cell response is indicated. (B) Diversity and magnitude of the HLA-E UL40 CD8 T-cell responses in KTR and HV. HLA-E UL40 CD8 T-cell responses appear in blue and colour intensity is proportional to the percentage of HLA-E UL40 CD8 T cells. (C) Classification of the HLA-E UL40 CD8 T-cell responses in HCMV + hosts according to possible recognition of self (orange), donor-specific allogeneic (green) or both (violet) (n = 31, 23 KTR and 8 HV). Grey boxes show HLA-I signal peptides which are not derived from the recipient, nor from the donor. Asterisks indicate peptides with underestimated information due to a lack of HLA-C genotyping.

Techniques Used: Isolation, Transplantation Assay, Ex Vivo, Flow Cytometry, Cytometry, Derivative Assay

Frequency of unconventional HLA-E UL40 CD8 T-cell responses compared to conventional HLA-A*02 pp65 CD8 T-cell responses in HCMV + kidney transplant recipients and healthy volunteers. PBMCs were isolated from freshly or prospectively harvested at M12 post-transplantation blood samples issued from healthy donors (HV) or from kidney transplant recipients (KTR), respectively. Ex vivo detection of HLA-E UL40 CD8 T and HLA-A*02 pp65 CD8 T cells was performed using flow cytometry by selecting CD3 + CD8α + TCRγδ - tetramer + cells on PBMCs. Detection threshold was 0.1% of total CD8 αβT cells and kidney transplant recipients and healthy volunteers bearing ≥0.1% of HLA-E UL40 CD8 T cells (in blue) or ≥0.1% HLA-A*02 pp65 CD8 T cells (in red) were considered as positive. Detection of both types of CD8 T-cell responses are indicated in violet. Absence of detection is shown in light grey in HCMV - recipients and dark grey for HCMV + hosts. Data shown are the number of individuals that display anti-HCMV CD8 T-cell responses. Frequencies of the CD8 T-cell subsets were calculated among subgroups for all (total), non HLA-A*02 and HLA-A*02 individuals and expressed as percentages (%).
Figure Legend Snippet: Frequency of unconventional HLA-E UL40 CD8 T-cell responses compared to conventional HLA-A*02 pp65 CD8 T-cell responses in HCMV + kidney transplant recipients and healthy volunteers. PBMCs were isolated from freshly or prospectively harvested at M12 post-transplantation blood samples issued from healthy donors (HV) or from kidney transplant recipients (KTR), respectively. Ex vivo detection of HLA-E UL40 CD8 T and HLA-A*02 pp65 CD8 T cells was performed using flow cytometry by selecting CD3 + CD8α + TCRγδ - tetramer + cells on PBMCs. Detection threshold was 0.1% of total CD8 αβT cells and kidney transplant recipients and healthy volunteers bearing ≥0.1% of HLA-E UL40 CD8 T cells (in blue) or ≥0.1% HLA-A*02 pp65 CD8 T cells (in red) were considered as positive. Detection of both types of CD8 T-cell responses are indicated in violet. Absence of detection is shown in light grey in HCMV - recipients and dark grey for HCMV + hosts. Data shown are the number of individuals that display anti-HCMV CD8 T-cell responses. Frequencies of the CD8 T-cell subsets were calculated among subgroups for all (total), non HLA-A*02 and HLA-A*02 individuals and expressed as percentages (%).

Techniques Used: Isolation, Transplantation Assay, Ex Vivo, Flow Cytometry, Cytometry

Diversity of HLA-E UL40 CD8 TCR Vβ repertoire and peptide recognition. HLA-E UL40 CD8 T cells were sorted from PBMCs for 5 KTR (#104, #105, #107, #108 and #109) and amplified in vitro . (A) Diversity of the HLA-E UL40 -specific TCR Vβ repertoire. Percentages indicate the ratio of individual Vβ chains by the total repertoire for each patient. (B) Specificity of peptide recognition toward UL40 and HLA-I peptides. HLA-E UL40 -specific CD8 T cells were stimulated for 5h with one of the eleven HLA-E UL40 tetramers and the percentage of TNF-producing cells among total CD8α cells was determined. Red arrows indicate the HCMV UL40 peptide provided by the infecting strain. Recognition of HLA-Ia signal peptides derived from autologous (orange bars), transplant-specific allogeneic (green bars) or both (purple bars) is shown. Grey bars indicate recognition of peptides that do not match with UL40 from the infecting strain or with HLA-Ia signal peptides not expressed by donors or hosts. Asterisks indicate samples with missing data for HLA-C genotype.
Figure Legend Snippet: Diversity of HLA-E UL40 CD8 TCR Vβ repertoire and peptide recognition. HLA-E UL40 CD8 T cells were sorted from PBMCs for 5 KTR (#104, #105, #107, #108 and #109) and amplified in vitro . (A) Diversity of the HLA-E UL40 -specific TCR Vβ repertoire. Percentages indicate the ratio of individual Vβ chains by the total repertoire for each patient. (B) Specificity of peptide recognition toward UL40 and HLA-I peptides. HLA-E UL40 -specific CD8 T cells were stimulated for 5h with one of the eleven HLA-E UL40 tetramers and the percentage of TNF-producing cells among total CD8α cells was determined. Red arrows indicate the HCMV UL40 peptide provided by the infecting strain. Recognition of HLA-Ia signal peptides derived from autologous (orange bars), transplant-specific allogeneic (green bars) or both (purple bars) is shown. Grey bars indicate recognition of peptides that do not match with UL40 from the infecting strain or with HLA-Ia signal peptides not expressed by donors or hosts. Asterisks indicate samples with missing data for HLA-C genotype.

Techniques Used: Amplification, In Vitro, IA, Derivative Assay

16) Product Images from "NK Cell Activation in Human Hantavirus Infection Explained by Virus-Induced IL-15/IL15Rα Expression"

Article Title: NK Cell Activation in Human Hantavirus Infection Explained by Virus-Induced IL-15/IL15Rα Expression

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004521

CD56 dim NK cells are highly activated in HFRS patients. (A) PBMC from 16 PUUV-infected HFRS patients were collected in the early acute (median d6) and convalescence phase (d60). From 8 HFRS patients additional samples were collected at the indicated time-points and up to day 450. Analyses of NK cell phenotype shown in 1B and 1D–G were performed with samples from patients in the early acute (median d6) and early convalescent phase (d60) of HFRS. (B) Frequencies of CD69-positive CD56 bright and CD56 dim NK cells in early acute and convalescent (d60) HFRS infection (n = 8). (** p≤0.01, paired t -test). (C) Frequencies of CD69-positive CD56 dim NK cells from HFRS patients (n = 8) from early acute to convalescence phases are depicted. Values shown are mean (+/− SD). (D and E) Expression levels of activating NK cell receptors on CD56 bright and CD56 dim NK cells in acute and convalescent phases (D) Representative staining for NKG2D, 2B4, NKp30 and NKp46 on CD56 bright and CD56 dim NK cells during acute HFRS infection (black) and convalescence (white). Isotype (grey). (E) Expression levels (MFI) of NKG2D, 2B4, NKp30 and NKp46 on CD56 bright and CD56 dim NK cells in HFRS patients (n = 14–16) in acute (black) and convalescent phases (white). (*** p≤0.001, paired t -test). (F and G) Levels of intracellular cytotoxic effector molecules in CD56 bright and CD56 dim NK cells in acute and convalescent phases. (F) Representative intracellular staining for granzyme B and perforin in CD56 bright and CD56 dim NK cells in acute HFRS infection (black) and convalescence (white). Isotype (grey). (G) Expression levels (MFI) of intracellular granzyme B and perforin in CD56 bright and CD56 dim NK cells of HFRS patients (n = 14–16) in acute (black) and convalescent phases of infection (white). (*** p≤0.001, * p≤0.05, paired t -test).
Figure Legend Snippet: CD56 dim NK cells are highly activated in HFRS patients. (A) PBMC from 16 PUUV-infected HFRS patients were collected in the early acute (median d6) and convalescence phase (d60). From 8 HFRS patients additional samples were collected at the indicated time-points and up to day 450. Analyses of NK cell phenotype shown in 1B and 1D–G were performed with samples from patients in the early acute (median d6) and early convalescent phase (d60) of HFRS. (B) Frequencies of CD69-positive CD56 bright and CD56 dim NK cells in early acute and convalescent (d60) HFRS infection (n = 8). (** p≤0.01, paired t -test). (C) Frequencies of CD69-positive CD56 dim NK cells from HFRS patients (n = 8) from early acute to convalescence phases are depicted. Values shown are mean (+/− SD). (D and E) Expression levels of activating NK cell receptors on CD56 bright and CD56 dim NK cells in acute and convalescent phases (D) Representative staining for NKG2D, 2B4, NKp30 and NKp46 on CD56 bright and CD56 dim NK cells during acute HFRS infection (black) and convalescence (white). Isotype (grey). (E) Expression levels (MFI) of NKG2D, 2B4, NKp30 and NKp46 on CD56 bright and CD56 dim NK cells in HFRS patients (n = 14–16) in acute (black) and convalescent phases (white). (*** p≤0.001, paired t -test). (F and G) Levels of intracellular cytotoxic effector molecules in CD56 bright and CD56 dim NK cells in acute and convalescent phases. (F) Representative intracellular staining for granzyme B and perforin in CD56 bright and CD56 dim NK cells in acute HFRS infection (black) and convalescence (white). Isotype (grey). (G) Expression levels (MFI) of intracellular granzyme B and perforin in CD56 bright and CD56 dim NK cells of HFRS patients (n = 14–16) in acute (black) and convalescent phases of infection (white). (*** p≤0.001, * p≤0.05, paired t -test).

Techniques Used: Infection, Expressing, Staining

17) Product Images from "Cholesterol crystals induce complement-dependent inflammasome activation and cytokine release"

Article Title: Cholesterol crystals induce complement-dependent inflammasome activation and cytokine release

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

doi: 10.4049/jimmunol.1302484

Combining C5a and TNF prime PBMC and monocytes for CC-induced IL-1β
Figure Legend Snippet: Combining C5a and TNF prime PBMC and monocytes for CC-induced IL-1β

Techniques Used:

18) Product Images from "Class I HLA haplotypes form two schools that educate NK cells in different ways"

Article Title: Class I HLA haplotypes form two schools that educate NK cells in different ways

Journal: Science immunology

doi: 10.1126/sciimmunol.aag1672

-21 HLA-B dimorphism modulates NK cell effector function A. PBMC were cultured for 6 hours in an ADCC assay with Raji cells (E:T = 10:1) coated with with anti-CD20 Ab or murine IgG (IgG) (range: 0.32–10µg/ml) and the CD3 − CD56 dim NK cells assayed for surface expression of CD107a. B. PBMC were cultured for 18 hours with cytokines and the frequency of IFN-γ + CD3 − CD56 dim NK cells determined. Cytokines were IL-12, IL-15, or IL-12 and IL-15 (range: 0.001–100ng/ml). C and D. PBMC were cultured with Raji cells for 18 hours and then with K562 cells for 6 hours (E:T = 10:1). CD3 − CD56 dim NK cells were assayed for CD107a (C) or IFN-γ (D). PBMC from all 60 donors were included in these analyses. M/M donors ( dark blue boxes ) M/T donors ( light blue ) and T/T donors ( yellow ). Statistical significance (p values) derives from two-tailed unpaired Mann Whitney tests (mean ± SE).
Figure Legend Snippet: -21 HLA-B dimorphism modulates NK cell effector function A. PBMC were cultured for 6 hours in an ADCC assay with Raji cells (E:T = 10:1) coated with with anti-CD20 Ab or murine IgG (IgG) (range: 0.32–10µg/ml) and the CD3 − CD56 dim NK cells assayed for surface expression of CD107a. B. PBMC were cultured for 18 hours with cytokines and the frequency of IFN-γ + CD3 − CD56 dim NK cells determined. Cytokines were IL-12, IL-15, or IL-12 and IL-15 (range: 0.001–100ng/ml). C and D. PBMC were cultured with Raji cells for 18 hours and then with K562 cells for 6 hours (E:T = 10:1). CD3 − CD56 dim NK cells were assayed for CD107a (C) or IFN-γ (D). PBMC from all 60 donors were included in these analyses. M/M donors ( dark blue boxes ) M/T donors ( light blue ) and T/T donors ( yellow ). Statistical significance (p values) derives from two-tailed unpaired Mann Whitney tests (mean ± SE).

Techniques Used: Cell Culture, ADCC Assay, Expressing, Two Tailed Test, MANN-WHITNEY

19) Product Images from "Expression of CXCL12 receptors in B cells from Mexican Mestizos patients with systemic lupus erythematosus"

Article Title: Expression of CXCL12 receptors in B cells from Mexican Mestizos patients with systemic lupus erythematosus

Journal: Journal of Translational Medicine

doi: 10.1186/1479-5876-10-251

Intracellular localization of CXCR4 and CXCR7 in SLE B cells. (A) Total pools of CXCR4 and CXCR7 were simultaneously detected by flow-cytometric analysis in CD19 + -gated PBMC from healthy and SLE subjects. Staining was performed on fixed and permeabilized leukocytes with both 9C4 (PE) and 12G5 (APC) mAbs. Quadrants were set on controls stained with the corresponding isotype control. Representative dot-plots demonstrating coexpression of CXCR4 and CXCR7 in total B cells from a healthy individual and two patients with either inactive or active SLE are shown. (B) The proportions (mean ± SEM) of total (CD19 + ) B cells, naive (CD19 + CD27 - ) B cells, memory (CD19 + CD27 + ) B cells and PC (CD19 low CD27 high ) expressing CXCR4 and/or CXCR7 in healthy and SLE subjects are given. * P
Figure Legend Snippet: Intracellular localization of CXCR4 and CXCR7 in SLE B cells. (A) Total pools of CXCR4 and CXCR7 were simultaneously detected by flow-cytometric analysis in CD19 + -gated PBMC from healthy and SLE subjects. Staining was performed on fixed and permeabilized leukocytes with both 9C4 (PE) and 12G5 (APC) mAbs. Quadrants were set on controls stained with the corresponding isotype control. Representative dot-plots demonstrating coexpression of CXCR4 and CXCR7 in total B cells from a healthy individual and two patients with either inactive or active SLE are shown. (B) The proportions (mean ± SEM) of total (CD19 + ) B cells, naive (CD19 + CD27 - ) B cells, memory (CD19 + CD27 + ) B cells and PC (CD19 low CD27 high ) expressing CXCR4 and/or CXCR7 in healthy and SLE subjects are given. * P

Techniques Used: Flow Cytometry, Staining, Expressing

Decreased levels of CXCR4 and CXCR7 mRNAs in active SLE leukocytes. (A and B) The relative levels of CXCR4 (A) and CXCR7 (B) transcripts in PBMC from SLE patients, distributed according to disease activity ( i.e. inactive versus active), were compared by real-time PCR with those from healthy individuals. Each individual sample has been run in triplicate. Results are expressed as CXCR4 / β-actin or CXCR7 / β-actin AU ratio and presented as box plots with median values, 25 th and 75 th quartile and the range of values. Kruskal-Wallis H test and associated P values are indicated. * P
Figure Legend Snippet: Decreased levels of CXCR4 and CXCR7 mRNAs in active SLE leukocytes. (A and B) The relative levels of CXCR4 (A) and CXCR7 (B) transcripts in PBMC from SLE patients, distributed according to disease activity ( i.e. inactive versus active), were compared by real-time PCR with those from healthy individuals. Each individual sample has been run in triplicate. Results are expressed as CXCR4 / β-actin or CXCR7 / β-actin AU ratio and presented as box plots with median values, 25 th and 75 th quartile and the range of values. Kruskal-Wallis H test and associated P values are indicated. * P

Techniques Used: Activity Assay, Real-time Polymerase Chain Reaction

Loss of CXCR4 expression on SLE B cells. (A) Membrane expression of CXCR4 on CD19 + -gated PBMC from healthy and SLE subjects was determined by flow cytometry using the APC-conjugated 12G5 (empty histograms) or isotype control (filled histogram) mAb. Displayed data are representative plots of the mean fluorescence intensity (MFI) of CXCR4 at the surface of total B cells from a healthy individual and two patients with either inactive or active SLE. (B) The percentage of total (CD19 + ) B cells, naive (CD19 + CD27 - ) B cells, memory (CD19 + CD27 + ) B cells and PC (CD19 low CD27 high ) expressing CXCR4 in healthy and SLE subjects are given. Box plots show the median values, 25 th and 75 th quartile and the range of values. Kruskal-Wallis H test and associated P values are indicated. *** P
Figure Legend Snippet: Loss of CXCR4 expression on SLE B cells. (A) Membrane expression of CXCR4 on CD19 + -gated PBMC from healthy and SLE subjects was determined by flow cytometry using the APC-conjugated 12G5 (empty histograms) or isotype control (filled histogram) mAb. Displayed data are representative plots of the mean fluorescence intensity (MFI) of CXCR4 at the surface of total B cells from a healthy individual and two patients with either inactive or active SLE. (B) The percentage of total (CD19 + ) B cells, naive (CD19 + CD27 - ) B cells, memory (CD19 + CD27 + ) B cells and PC (CD19 low CD27 high ) expressing CXCR4 in healthy and SLE subjects are given. Box plots show the median values, 25 th and 75 th quartile and the range of values. Kruskal-Wallis H test and associated P values are indicated. *** P

Techniques Used: Expressing, Flow Cytometry, Cytometry, Fluorescence

Altered CXCR7 expression on SLE B cells. (A) Surface expression of CXCR7 on CD19 + -gated PBMC from healthy and SLE subjects was determined by flow cytometry using the unconjugated 9C4 (empty histograms) or isotype control (filled histograms) mAb followed by a PE-conjugated goat anti-mouse F(ab’) 2 Ab. CXCR7 -transduced ( versus parental) HEK 293 T (HEK) cells were used as a positive control for staining. Displayed data are representative plots of the MFI of CXCR7 at the surface of total B cells from a healthy individual and two patients with either inactive or active SLE (left panel) or of HEK cells transduced with CXCR7 or left untransduced (right panel). (B) The percentage of total (CD19 + ) B cells, naive (CD19 + CD27 - ) B cells, memory (CD19 + CD27 + ) B cells and PC (CD19 low CD27 high ) expressing CXCR7 in healthy and SLE subjects are shown. Box plots show the median values, 25 th and 75 th quartile and the range of values. Kruskal-Wallis H test and associated P values are indicated. * P
Figure Legend Snippet: Altered CXCR7 expression on SLE B cells. (A) Surface expression of CXCR7 on CD19 + -gated PBMC from healthy and SLE subjects was determined by flow cytometry using the unconjugated 9C4 (empty histograms) or isotype control (filled histograms) mAb followed by a PE-conjugated goat anti-mouse F(ab’) 2 Ab. CXCR7 -transduced ( versus parental) HEK 293 T (HEK) cells were used as a positive control for staining. Displayed data are representative plots of the MFI of CXCR7 at the surface of total B cells from a healthy individual and two patients with either inactive or active SLE (left panel) or of HEK cells transduced with CXCR7 or left untransduced (right panel). (B) The percentage of total (CD19 + ) B cells, naive (CD19 + CD27 - ) B cells, memory (CD19 + CD27 + ) B cells and PC (CD19 low CD27 high ) expressing CXCR7 in healthy and SLE subjects are shown. Box plots show the median values, 25 th and 75 th quartile and the range of values. Kruskal-Wallis H test and associated P values are indicated. * P

Techniques Used: Expressing, Flow Cytometry, Cytometry, Positive Control, Staining, Transduction

Loss of CXCL12 responsiveness in SLE B cells. (A and B) PBMC from independent healthy individuals (n = 9) and patients (SLE, n = 14), with either inactive (n = 7) or active (n = 7) disease, were tested for their ability to migrate in response to the indicated concentration (A) of chemokine or 30 nM of CXCL12 (B) . Transmigrated cells recovered in the lower chamber were stained with mAbs specific for CD19, CD20, CD38 and CD27 antigens and counted by flow cytometry. Inhibition of cell migration by AMD3100 added in both chambers is shown. Results (mean ± SEM) are from 4 independent experiments and expressed as the percentage of input total (gated CD19 + ) B cells (A and B, upper left), naive (gated CD19 + CD20 + CD27 - CD38 - ) B cells (B, upper right), memory (gated CD19 + CD20 + CD27 + CD38 - ) B cells (B, lower left) or PC (gated CD19 low CD20 low CD27 high CD38 high ) (B, lower right) that migrated to the lower chamber. * P
Figure Legend Snippet: Loss of CXCL12 responsiveness in SLE B cells. (A and B) PBMC from independent healthy individuals (n = 9) and patients (SLE, n = 14), with either inactive (n = 7) or active (n = 7) disease, were tested for their ability to migrate in response to the indicated concentration (A) of chemokine or 30 nM of CXCL12 (B) . Transmigrated cells recovered in the lower chamber were stained with mAbs specific for CD19, CD20, CD38 and CD27 antigens and counted by flow cytometry. Inhibition of cell migration by AMD3100 added in both chambers is shown. Results (mean ± SEM) are from 4 independent experiments and expressed as the percentage of input total (gated CD19 + ) B cells (A and B, upper left), naive (gated CD19 + CD20 + CD27 - CD38 - ) B cells (B, upper right), memory (gated CD19 + CD20 + CD27 + CD38 - ) B cells (B, lower left) or PC (gated CD19 low CD20 low CD27 high CD38 high ) (B, lower right) that migrated to the lower chamber. * P

Techniques Used: Concentration Assay, Staining, Flow Cytometry, Cytometry, Inhibition, Migration

Increased frequency of circulating plasma cells in SLE patients. (A) Double staining with CD19 and CD27 was performed on PBMC to delineate naive (CD19 + CD27 - ) B cells, memory (CD19 + CD27 + ) B cells and plasma (PC, CD19 low CD27 high ) cells. Thresholds and fluorescence gates used for the statistical evaluation of CD27 - , CD27 + and CD27 high B lymphocytes are indicated as well as the corresponding frequency of these subsets among total B cells. Expression of CD27 on CD19 + peripheral B cells is shown for a representative healthy blood donor and a patient with active SLE. (B) Comparison of the frequencies of total B cells among PBMC and of the different B-cell subpopulations from SLE patients (n = 41), distributed according to disease activity, i.e. inactive (n = 17) versus active (n = 24), and from healthy individuals (n = 45). The proportions were determined by flow-cytometric analysis as shown in A . Results are depicted as box plots, with median (horizontal line within each box) and 10 th , 25 th , 75 th , and 90 th percentiles (bottom bar, bottom of box, top of box, and top bar, respectively). Kruskal-Wallis H test and associated P values are indicated. * P
Figure Legend Snippet: Increased frequency of circulating plasma cells in SLE patients. (A) Double staining with CD19 and CD27 was performed on PBMC to delineate naive (CD19 + CD27 - ) B cells, memory (CD19 + CD27 + ) B cells and plasma (PC, CD19 low CD27 high ) cells. Thresholds and fluorescence gates used for the statistical evaluation of CD27 - , CD27 + and CD27 high B lymphocytes are indicated as well as the corresponding frequency of these subsets among total B cells. Expression of CD27 on CD19 + peripheral B cells is shown for a representative healthy blood donor and a patient with active SLE. (B) Comparison of the frequencies of total B cells among PBMC and of the different B-cell subpopulations from SLE patients (n = 41), distributed according to disease activity, i.e. inactive (n = 17) versus active (n = 24), and from healthy individuals (n = 45). The proportions were determined by flow-cytometric analysis as shown in A . Results are depicted as box plots, with median (horizontal line within each box) and 10 th , 25 th , 75 th , and 90 th percentiles (bottom bar, bottom of box, top of box, and top bar, respectively). Kruskal-Wallis H test and associated P values are indicated. * P

Techniques Used: Double Staining, Fluorescence, Expressing, Activity Assay, Flow Cytometry

20) Product Images from "Suppressor of Cytokine Signaling-1 (SOCS1) Inhibits Lymphocyte Recruitment into the Retina and Protects SOCS1 Transgenic Rats and Mice from Ocular Inflammation"

Article Title: Suppressor of Cytokine Signaling-1 (SOCS1) Inhibits Lymphocyte Recruitment into the Retina and Protects SOCS1 Transgenic Rats and Mice from Ocular Inflammation

Journal: Investigative Ophthalmology & Visual Science

doi: 10.1167/iovs.11-7688

SOCS1 inhibits recruitment of inflammatory cells into the retina. ( A ) Freshly isolated PBMC or LN cells from WT or SOCS1-Tg mice at early stage of EAU (day 14 postimmunization) were analyzed by the intracellular cytokine assay. CD4 + T cells were gated
Figure Legend Snippet: SOCS1 inhibits recruitment of inflammatory cells into the retina. ( A ) Freshly isolated PBMC or LN cells from WT or SOCS1-Tg mice at early stage of EAU (day 14 postimmunization) were analyzed by the intracellular cytokine assay. CD4 + T cells were gated

Techniques Used: Isolation, Mouse Assay, Cytokine Assay

SOCS1 inhibits expression of proinflammatory molecules that mediate intraocular inflammation. ( A ) Freshly isolated T cells from spleen, LNs, and PBMCs of WT mice with EAU were analyzed by the intracellular cytokine assay. Numbers in quadrants indicate
Figure Legend Snippet: SOCS1 inhibits expression of proinflammatory molecules that mediate intraocular inflammation. ( A ) Freshly isolated T cells from spleen, LNs, and PBMCs of WT mice with EAU were analyzed by the intracellular cytokine assay. Numbers in quadrants indicate

Techniques Used: Expressing, Isolation, Mouse Assay, Cytokine Assay

21) Product Images from "CISH promoter polymorphism effects on T cell cytokine receptor signaling and type 1 diabetes susceptibility"

Article Title: CISH promoter polymorphism effects on T cell cytokine receptor signaling and type 1 diabetes susceptibility

Journal: Molecular and Cellular Pediatrics

doi: 10.1186/s40348-018-0080-7

Cytokine induced CIS mRNA expression and STAT5 phosphorylation according to CISH haplotypes. a CIS mRNA expression of PBMCs from healthy adult donors with distinct haplotypes (circles: homozygous H1 carriers (H1/H1), triangles: H2 carriers (H1/H2, H2/H2), squares: H3 carriers (H1/H3)) treated with IL-2 (left graph) or IL-7 (right graph) for 1 and 2 h. CIS mRNA levels—calculated in comparison to the housekeeping gene GAPDH—are normalized against time point 0 for each donor. Median and IQR of 2 −ΔΔCT values are indicated. p value for effect of IL-2/IL-7 stimulation on CIS mRNA levels considering all genotypes (Friedman test) is indicated as *** for p
Figure Legend Snippet: Cytokine induced CIS mRNA expression and STAT5 phosphorylation according to CISH haplotypes. a CIS mRNA expression of PBMCs from healthy adult donors with distinct haplotypes (circles: homozygous H1 carriers (H1/H1), triangles: H2 carriers (H1/H2, H2/H2), squares: H3 carriers (H1/H3)) treated with IL-2 (left graph) or IL-7 (right graph) for 1 and 2 h. CIS mRNA levels—calculated in comparison to the housekeeping gene GAPDH—are normalized against time point 0 for each donor. Median and IQR of 2 −ΔΔCT values are indicated. p value for effect of IL-2/IL-7 stimulation on CIS mRNA levels considering all genotypes (Friedman test) is indicated as *** for p

Techniques Used: Expressing, Chromogenic In Situ Hybridization

22) Product Images from "Urothelial bladder cancer may suppress perforin expression in CD8+ T cells by an ICAM-1/TGFβ2 mediated pathway"

Article Title: Urothelial bladder cancer may suppress perforin expression in CD8+ T cells by an ICAM-1/TGFβ2 mediated pathway

Journal: PLoS ONE

doi: 10.1371/journal.pone.0200079

Tumor adjacent lymph nodes had lower CD8 + T cells cytotoxicity. Patients’ samples were received from trans-urethral resection of the bladder (TUR-B) or cystectomies (n = 27). ( A ) Comparison of CD8 + T cells distribution was done between peripheral blood mononuclear cells (PBMC), sentinel nodes (SN), and tumor after immunophenotyping by flow cytometry. CD8 + T cells distribution was calculated from CD3 + lymphocytes population. The data are means with the error bars indicating SEM. One-way ANOVA was used as the statistical test. ( B ) The cytotoxic phenotype of CD8 + T cells in different tissues from UBC patients was analyzed by flow cytometry. The frequencies of granzyme B expressing CD8 + T cells were compared among PBMC, SN, and tumor. ( C ) Same as in (B) but the analysis was done on perforin expressing CD8 + T cells. The black middle lines indicate median. Kruskal-Wallis was used as the statistical test. * p
Figure Legend Snippet: Tumor adjacent lymph nodes had lower CD8 + T cells cytotoxicity. Patients’ samples were received from trans-urethral resection of the bladder (TUR-B) or cystectomies (n = 27). ( A ) Comparison of CD8 + T cells distribution was done between peripheral blood mononuclear cells (PBMC), sentinel nodes (SN), and tumor after immunophenotyping by flow cytometry. CD8 + T cells distribution was calculated from CD3 + lymphocytes population. The data are means with the error bars indicating SEM. One-way ANOVA was used as the statistical test. ( B ) The cytotoxic phenotype of CD8 + T cells in different tissues from UBC patients was analyzed by flow cytometry. The frequencies of granzyme B expressing CD8 + T cells were compared among PBMC, SN, and tumor. ( C ) Same as in (B) but the analysis was done on perforin expressing CD8 + T cells. The black middle lines indicate median. Kruskal-Wallis was used as the statistical test. * p

Techniques Used: Flow Cytometry, Cytometry, Expressing

Production and secretion of perforin in SN CD8 + T cells are low after in vitro reactivation. Lymphocytes isolated from peripheral blood (PBMC) and sentinel node (SN) were cultured for seven days with addition of autologous tumor homogenate. ( A ) Flow cytometry was done to phenotype the co-expression in CD8 + T cells from PBMC and SN before and after reactivation. The results were shown in dot plots and gated based on isotype control. The frequency of granzyme B and perforin expression was counted out of CD8 + T cells. Dot plots showed data from a representative cystectomized patient. ( B ) Intracellular perforin was measured by Median Fluorescence Intensity (MFI) post 7-day culture using flow cytometry from (A). The data are means with error bars indicating SEM. Mann-Whitney was used as the statistical test. ( C ) The concentrations (pg/ml) of secreted granzyme B and perforin after seven days of culture were analyzed by ELISA and compared between in vitro culture supernatants of PBMC and SN. The data are means with error bars indicating SEM. Mann-Whitney was used as the statistical test. *p
Figure Legend Snippet: Production and secretion of perforin in SN CD8 + T cells are low after in vitro reactivation. Lymphocytes isolated from peripheral blood (PBMC) and sentinel node (SN) were cultured for seven days with addition of autologous tumor homogenate. ( A ) Flow cytometry was done to phenotype the co-expression in CD8 + T cells from PBMC and SN before and after reactivation. The results were shown in dot plots and gated based on isotype control. The frequency of granzyme B and perforin expression was counted out of CD8 + T cells. Dot plots showed data from a representative cystectomized patient. ( B ) Intracellular perforin was measured by Median Fluorescence Intensity (MFI) post 7-day culture using flow cytometry from (A). The data are means with error bars indicating SEM. Mann-Whitney was used as the statistical test. ( C ) The concentrations (pg/ml) of secreted granzyme B and perforin after seven days of culture were analyzed by ELISA and compared between in vitro culture supernatants of PBMC and SN. The data are means with error bars indicating SEM. Mann-Whitney was used as the statistical test. *p

Techniques Used: In Vitro, Isolation, Cell Culture, Flow Cytometry, Cytometry, Expressing, Fluorescence, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay

Perforin deficiency in CD8 + T cells from sentinel nodes. ( A ) The expression of granzyme B and perforin in CD8 + T cells of different tissues were phenotyped by flow cytometry. The co-expression pattern in CD8 + T cells was shown in dot plots and gated for distinguishing between double and single expression of granzyme B and perforin. The gate was based on isotype control and the frequency of granzyme B and perforin expression was counted out of CD8 + T cells. Dot plots showed a representative data from a patient underwent transurethral resection of the bladder (TUR-B) and cystectomy. ( B ) The frequency of granzyme B + /perforin + CD8 + T cells from PBMC, SN, and tumor tissues was shown in graphs and was calculated out of CD8 + T cells (n = 27). ( C ) Same as in (B) but the analysis was done on granzyme B + /perforin - CD8 + T cells. The data are means with the error bars indicating SEM. Kruskal-Wallis was used as the statistical test. ( D ) The expression of gene responsible in encoding granzyme B ( GZMB ) and perforin ( PRF1 ) in CD8 + T cells isolated from PBMC, SN, and tumor (n = 6). RT-qPCR was done to analyze the gene expression followed by quantification using 2 -ΔΔCt method. The fold change was calculated in regards of PBMC as control, with RPII gene used as the housekeeping gene. The data are the means of Log 2 of fold change (2 -ΔΔCt ) with the error bars indicating SEM. Kruskal-Wallis was used as the statistical test on each gene. *p
Figure Legend Snippet: Perforin deficiency in CD8 + T cells from sentinel nodes. ( A ) The expression of granzyme B and perforin in CD8 + T cells of different tissues were phenotyped by flow cytometry. The co-expression pattern in CD8 + T cells was shown in dot plots and gated for distinguishing between double and single expression of granzyme B and perforin. The gate was based on isotype control and the frequency of granzyme B and perforin expression was counted out of CD8 + T cells. Dot plots showed a representative data from a patient underwent transurethral resection of the bladder (TUR-B) and cystectomy. ( B ) The frequency of granzyme B + /perforin + CD8 + T cells from PBMC, SN, and tumor tissues was shown in graphs and was calculated out of CD8 + T cells (n = 27). ( C ) Same as in (B) but the analysis was done on granzyme B + /perforin - CD8 + T cells. The data are means with the error bars indicating SEM. Kruskal-Wallis was used as the statistical test. ( D ) The expression of gene responsible in encoding granzyme B ( GZMB ) and perforin ( PRF1 ) in CD8 + T cells isolated from PBMC, SN, and tumor (n = 6). RT-qPCR was done to analyze the gene expression followed by quantification using 2 -ΔΔCt method. The fold change was calculated in regards of PBMC as control, with RPII gene used as the housekeeping gene. The data are the means of Log 2 of fold change (2 -ΔΔCt ) with the error bars indicating SEM. Kruskal-Wallis was used as the statistical test on each gene. *p

Techniques Used: Expressing, Flow Cytometry, Cytometry, Isolation, Quantitative RT-PCR

Sentinel node CD8 + T cells with perforin deficiency are exhausted Tc2 cells. ( A ) CD8 + T cells isolated from sentinel node (SN) were further phenotyped using flow cytometry to demonstrate the difference in T cells exhaustion markers expression (PD-1) and Tc1 transcription factor (T-bet) between granzyme B + /perforin − CD8 + T cells (green box) and granzyme B + /perforin + CD8 + T cells (red box). The expression of PD-1 and T-bet were shown in dot plots from a representative patient and gated based on isotype control. ( B ) The frequency of PD-1 and T-bet from (A) was calculated either out of granzyme B + /perforin − or granzyme B + /perforin + CD8 + T cells. The data are means with the error bars indicating SEM. Mann-Whitney was used as the statistical test. ( C ) The expression of T-bet, encoded by TBX21 gene, was compared among CD8 + T cells sorted from peripheral blood mononuclear cells (PBMC), sentinel node (SN), and tumor. mRNA was extracted from the sorted cells and the TBX21 gene expression was analyzed by RT-qPCR. The expression of TBX21 was quantified using 2 -ΔΔCt method and the fold change was calculated in regards of PBMC as control. RPII gene was used as housekeeping gene. The data are means with error bars indicating SEM. Kruskal-Wallis was used as the statistical test. ( D ) Same as in (C), but the analysis was done on GATA3 gene expression. ( E ) The frequency of naïve T cells (CD45RA + CCR7 + ), central memory T (T CM ) cells (CD45RA - CCR7 + ), effector memory T (T EM ) cells (CD45RA - CCR7 - ), and effector memory T with CD45RA expression (T EMRA ) cells (CD45RA + CCR7 - ) was calculated either out of granzyme B + /perforin − or granzyme B + /perforin + CD8 + T cells. The data are means with the error bars indicating SEM. Kruskal-Wallis was used as the statistical test. * p
Figure Legend Snippet: Sentinel node CD8 + T cells with perforin deficiency are exhausted Tc2 cells. ( A ) CD8 + T cells isolated from sentinel node (SN) were further phenotyped using flow cytometry to demonstrate the difference in T cells exhaustion markers expression (PD-1) and Tc1 transcription factor (T-bet) between granzyme B + /perforin − CD8 + T cells (green box) and granzyme B + /perforin + CD8 + T cells (red box). The expression of PD-1 and T-bet were shown in dot plots from a representative patient and gated based on isotype control. ( B ) The frequency of PD-1 and T-bet from (A) was calculated either out of granzyme B + /perforin − or granzyme B + /perforin + CD8 + T cells. The data are means with the error bars indicating SEM. Mann-Whitney was used as the statistical test. ( C ) The expression of T-bet, encoded by TBX21 gene, was compared among CD8 + T cells sorted from peripheral blood mononuclear cells (PBMC), sentinel node (SN), and tumor. mRNA was extracted from the sorted cells and the TBX21 gene expression was analyzed by RT-qPCR. The expression of TBX21 was quantified using 2 -ΔΔCt method and the fold change was calculated in regards of PBMC as control. RPII gene was used as housekeeping gene. The data are means with error bars indicating SEM. Kruskal-Wallis was used as the statistical test. ( D ) Same as in (C), but the analysis was done on GATA3 gene expression. ( E ) The frequency of naïve T cells (CD45RA + CCR7 + ), central memory T (T CM ) cells (CD45RA - CCR7 + ), effector memory T (T EM ) cells (CD45RA - CCR7 - ), and effector memory T with CD45RA expression (T EMRA ) cells (CD45RA + CCR7 - ) was calculated either out of granzyme B + /perforin − or granzyme B + /perforin + CD8 + T cells. The data are means with the error bars indicating SEM. Kruskal-Wallis was used as the statistical test. * p

Techniques Used: Isolation, Flow Cytometry, Cytometry, Expressing, MANN-WHITNEY, Quantitative RT-PCR

23) Product Images from "[18F]-Fluorodeoxyglucose Uptake in Lymphoid Tissue Serves as a Predictor of Disease Outcome in the Nonhuman Primate Model of Monkeypox Virus Infection"

Article Title: [18F]-Fluorodeoxyglucose Uptake in Lymphoid Tissue Serves as a Predictor of Disease Outcome in the Nonhuman Primate Model of Monkeypox Virus Infection

Journal: Journal of Virology

doi: 10.1128/JVI.00897-17

Proliferation of lymphocytes in LNs and PBMCs as measured by flow cytometry. (A) Ki-67 immunostaining of lymphocytes isolated from LNs of healthy animals on day −18 preexposure ( n = 2) and from LNs of surviving animals ( n = 4) on days 9 and 22 p.i. (B to D) Ki-67 immunostaining of CD4 + T cells (B), CD8 + T cells (C), and CD20 + B cells (D) in PBMCs in treated and untreated survivors versus moribund NHPs. Abbreviations: LN, lymph node; NHP, nonhuman primate; PBMCs, peripheral blood mononuclear cells; p.i., postinfection.
Figure Legend Snippet: Proliferation of lymphocytes in LNs and PBMCs as measured by flow cytometry. (A) Ki-67 immunostaining of lymphocytes isolated from LNs of healthy animals on day −18 preexposure ( n = 2) and from LNs of surviving animals ( n = 4) on days 9 and 22 p.i. (B to D) Ki-67 immunostaining of CD4 + T cells (B), CD8 + T cells (C), and CD20 + B cells (D) in PBMCs in treated and untreated survivors versus moribund NHPs. Abbreviations: LN, lymph node; NHP, nonhuman primate; PBMCs, peripheral blood mononuclear cells; p.i., postinfection.

Techniques Used: Flow Cytometry, Cytometry, Immunostaining, Isolation

24) Product Images from "A novel method for making human monoclonal antibodies"

Article Title: A novel method for making human monoclonal antibodies

Journal: Journal of Autoimmunity

doi: 10.1016/j.jaut.2010.05.001

Immortalization efficiency and clonality on isolated IgG + B cells or total PBMC. (a) Immortalization efficiency for isolated IgG + CD22 + cells using the improved B cell immortalization method was measured as the percentage of IgG- and growth-positive wells. IgG-producing immortalized B cell lines are shown 28 days after seeding for 3 healthy donors. (b) The mean percentage of immortalized B cell lines is shown after immortalization of isolated IgG + CD22 + cells and total PBMC using simultaneous B cell stimulation and infection with the addition of CpG2006, IL-2 or both after immortalization. Both IgG- and IgM-producing immortalized B cell lines are depicted for total PBMC. Results shown are the average of experiments in 2 healthy donors (donors 1 and 2 panel a). (c) Clonality of 34 randomly selected IgG + immortalized B cell lines was determined using B cell spectratyping for IgG + CD22 + cells and total PBMC. The percentage of monoclonal, biclonal and polyclonal immortalized B cell lines is shown.
Figure Legend Snippet: Immortalization efficiency and clonality on isolated IgG + B cells or total PBMC. (a) Immortalization efficiency for isolated IgG + CD22 + cells using the improved B cell immortalization method was measured as the percentage of IgG- and growth-positive wells. IgG-producing immortalized B cell lines are shown 28 days after seeding for 3 healthy donors. (b) The mean percentage of immortalized B cell lines is shown after immortalization of isolated IgG + CD22 + cells and total PBMC using simultaneous B cell stimulation and infection with the addition of CpG2006, IL-2 or both after immortalization. Both IgG- and IgM-producing immortalized B cell lines are depicted for total PBMC. Results shown are the average of experiments in 2 healthy donors (donors 1 and 2 panel a). (c) Clonality of 34 randomly selected IgG + immortalized B cell lines was determined using B cell spectratyping for IgG + CD22 + cells and total PBMC. The percentage of monoclonal, biclonal and polyclonal immortalized B cell lines is shown.

Techniques Used: Isolation, Cell Stimulation, Infection

B cell immortalization procedure. IgG + CD22 + B cells are isolated from PBMC by FACS sorting (1) and thereafter immortalized using simultaneous B cell stimulation and infection. B cells are cultured during 2 weeks in microtiter plates at 50 cells per well in the presence of 1 × 10 5 autologous irradiated feeder cells, 1 μg/ml CpG2006 and 30% v/v EBV-containing supernatant of the B95-8 cell line (2). After this immortalization phase, the cells are restimulated during 1 week with 1 μg/ml CpG2006 in combination with 50 U/ml IL-2 (3). The culture medium is then replaced and cultures are continued without the addition of stimuli for 1 week. Immortalization status is verified after 28 days of culture by performing dot blot analysis for antibody production and by light microscopic examination of cell growth (4).
Figure Legend Snippet: B cell immortalization procedure. IgG + CD22 + B cells are isolated from PBMC by FACS sorting (1) and thereafter immortalized using simultaneous B cell stimulation and infection. B cells are cultured during 2 weeks in microtiter plates at 50 cells per well in the presence of 1 × 10 5 autologous irradiated feeder cells, 1 μg/ml CpG2006 and 30% v/v EBV-containing supernatant of the B95-8 cell line (2). After this immortalization phase, the cells are restimulated during 1 week with 1 μg/ml CpG2006 in combination with 50 U/ml IL-2 (3). The culture medium is then replaced and cultures are continued without the addition of stimuli for 1 week. Immortalization status is verified after 28 days of culture by performing dot blot analysis for antibody production and by light microscopic examination of cell growth (4).

Techniques Used: Isolation, FACS, Cell Stimulation, Infection, Cell Culture, Irradiation, Dot Blot

25) Product Images from "IFN-? production in response to Tax 161-233, and frequency of CD4+ Foxp3+ and Lin− HLA-DRhigh CD123+ cells, discriminate HAM/TSP patients from asymptomatic HTLV-1-carriers in a Peruvian population"

Article Title: IFN-? production in response to Tax 161-233, and frequency of CD4+ Foxp3+ and Lin− HLA-DRhigh CD123+ cells, discriminate HAM/TSP patients from asymptomatic HTLV-1-carriers in a Peruvian population

Journal: Immunology

doi: 10.1111/j.1365-2567.2009.03082.x

Frequency of myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC). Data represent (a) the percentage of mDC, or (b) the percentage of pDC, in a Lin − HLA-DR + population from freshly isolated peripheral blood mononuclear cells (PBMC).
Figure Legend Snippet: Frequency of myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC). Data represent (a) the percentage of mDC, or (b) the percentage of pDC, in a Lin − HLA-DR + population from freshly isolated peripheral blood mononuclear cells (PBMC).

Techniques Used: Isolation

26) Product Images from "Disturbed Homeostasis and Multiple Signaling Defects in the Peripheral Blood B-Cell Compartment of Patients with Severe Chronic Sarcoidosis ▿"

Article Title: Disturbed Homeostasis and Multiple Signaling Defects in the Peripheral Blood B-Cell Compartment of Patients with Severe Chronic Sarcoidosis ▿

Journal: Clinical and Vaccine Immunology : CVI

doi: 10.1128/CVI.05118-11

Reduced levels of NF-κB/p65 in sarcoid B cells. Purified CD19 + cells isolated from PBMC of sarcoidosis patients and healthy controls by magnetic-activated cell sorting were lysed, and protein lysates were electrophoretically separated, transferred
Figure Legend Snippet: Reduced levels of NF-κB/p65 in sarcoid B cells. Purified CD19 + cells isolated from PBMC of sarcoidosis patients and healthy controls by magnetic-activated cell sorting were lysed, and protein lysates were electrophoretically separated, transferred

Techniques Used: Purification, Isolation, FACS

Decreased numbers and frequency of memory B cells in sarcoidosis patients. (A) Representative example of CD19 and CD27 expression on PBMC. The frequencies of naïve and memory B cells in sarcoidosis patients and healthy controls are compared. Results
Figure Legend Snippet: Decreased numbers and frequency of memory B cells in sarcoidosis patients. (A) Representative example of CD19 and CD27 expression on PBMC. The frequencies of naïve and memory B cells in sarcoidosis patients and healthy controls are compared. Results

Techniques Used: Expressing

Impaired activation, proliferation, and differentiation of sarcoid B cells. (A to C) CD19 + lymphocytes, purified from PBMC of sarcoid patients and healthy controls by magnetic-activated cell sorting, were incubated for 24 h with CD40L, CpG-ODN, IL-2,
Figure Legend Snippet: Impaired activation, proliferation, and differentiation of sarcoid B cells. (A to C) CD19 + lymphocytes, purified from PBMC of sarcoid patients and healthy controls by magnetic-activated cell sorting, were incubated for 24 h with CD40L, CpG-ODN, IL-2,

Techniques Used: Activation Assay, Purification, FACS, Incubation

Expression levels of costimulatory (CD19, CD21, and CD45) and coinhibitory (CD32, CD35, CD85J, and CD152) receptors in sarcoid B cells. CD19 + cells, purified from PBMC of sarcoid patients and healthy controls by magnetic-activated cell sorting, were either
Figure Legend Snippet: Expression levels of costimulatory (CD19, CD21, and CD45) and coinhibitory (CD32, CD35, CD85J, and CD152) receptors in sarcoid B cells. CD19 + cells, purified from PBMC of sarcoid patients and healthy controls by magnetic-activated cell sorting, were either

Techniques Used: Expressing, Purification, FACS

27) Product Images from "A new peptide vaccine OCV-501: in vitro pharmacology and phase 1 study in patients with acute myeloid leukemia"

Article Title: A new peptide vaccine OCV-501: in vitro pharmacology and phase 1 study in patients with acute myeloid leukemia

Journal: Cancer Immunology, Immunotherapy

doi: 10.1007/s00262-017-1981-3

Efficacy pharmacology studies of OCV-501 using PBMC from healthy donor. Th1: Type 1 T-helper, WT1: Wilms’ tumor 1. Induction of OCV-501-specific Th1 cells in PBMC ( a ). PBMC were cultured with OCV-501 ( filled circle ) or solvent ( circle ) (data are expressed as mean ± SE: n = 20, * P = 0.0058: main effect in mixed model for repeated measures method, crossover type). The HLA class II-restriction of OCV-501-derived antigen stimulation was evaluated using OCV-501-specific T cells induced in PBMC by blockade with anti-HLA class II antibody ( b ) (data are expressed as mean ± SD, triplicates). Dose-dependence of OCV-501 activation of specific T cells ( c ) (data are expressed as mean ± SE: n = 20, * P
Figure Legend Snippet: Efficacy pharmacology studies of OCV-501 using PBMC from healthy donor. Th1: Type 1 T-helper, WT1: Wilms’ tumor 1. Induction of OCV-501-specific Th1 cells in PBMC ( a ). PBMC were cultured with OCV-501 ( filled circle ) or solvent ( circle ) (data are expressed as mean ± SE: n = 20, * P = 0.0058: main effect in mixed model for repeated measures method, crossover type). The HLA class II-restriction of OCV-501-derived antigen stimulation was evaluated using OCV-501-specific T cells induced in PBMC by blockade with anti-HLA class II antibody ( b ) (data are expressed as mean ± SD, triplicates). Dose-dependence of OCV-501 activation of specific T cells ( c ) (data are expressed as mean ± SE: n = 20, * P

Techniques Used: Wilms Tumor Assay, Cell Culture, Derivative Assay, Activation Assay

28) Product Images from "Retinoic acid-producing, ex-vivo-generated human tolerogenic dendritic cells induce the proliferation of immunosuppressive B lymphocytes"

Article Title: Retinoic acid-producing, ex-vivo-generated human tolerogenic dendritic cells induce the proliferation of immunosuppressive B lymphocytes

Journal: Clinical and Experimental Immunology

doi: 10.1111/cei.12177

Peripheral blood mononuclear cells (PBMC) exposure to rheumatoid arthritis (RA) in vitro results in increased frequency of CD19 + CD24 + CD38 + regulatory B cells (B regs ). (a) Freshly collected PBMC from unrelated healthy adults were treated with all-
Figure Legend Snippet: Peripheral blood mononuclear cells (PBMC) exposure to rheumatoid arthritis (RA) in vitro results in increased frequency of CD19 + CD24 + CD38 + regulatory B cells (B regs ). (a) Freshly collected PBMC from unrelated healthy adults were treated with all-

Techniques Used: In Vitro

Frequency and suppressive character in vitro of CD19 + CD24 + B cells from freshly collected peripheral blood mononuclear cells (PBMC) from normal healthy humans. (a) The graph summarizes the median and the range of the % CD19 + CD24 + CD27 + CD38 + B cells [human
Figure Legend Snippet: Frequency and suppressive character in vitro of CD19 + CD24 + B cells from freshly collected peripheral blood mononuclear cells (PBMC) from normal healthy humans. (a) The graph summarizes the median and the range of the % CD19 + CD24 + CD27 + CD38 + B cells [human

Techniques Used: In Vitro

29) Product Images from "Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient"

Article Title: Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient

Journal: BMC Immunology

doi: 10.1186/s12865-015-0113-0

Signal scatter plots comparing the intensities of gene expressions on microarray chips within Ficoll and CPT-derived PBMC and their immune cell subsets for each healthy donor tested. The expression signals of Ficoll-derived cells were plotted against the expression signals of CPT-derived cells for each cell type and donor. A line of perfect correlation is indicated in all plots. Pearson’s correlation coefficients between each pair of samples are shown at the bottom right of each plot. The data showed that the pattern of gene expression was very similar between Ficoll and CPT-derived cell types, as indicated by the high correlation coefficients
Figure Legend Snippet: Signal scatter plots comparing the intensities of gene expressions on microarray chips within Ficoll and CPT-derived PBMC and their immune cell subsets for each healthy donor tested. The expression signals of Ficoll-derived cells were plotted against the expression signals of CPT-derived cells for each cell type and donor. A line of perfect correlation is indicated in all plots. Pearson’s correlation coefficients between each pair of samples are shown at the bottom right of each plot. The data showed that the pattern of gene expression was very similar between Ficoll and CPT-derived cell types, as indicated by the high correlation coefficients

Techniques Used: Microarray, Cycling Probe Technology, Derivative Assay, Expressing

Yield and viability of immune cell subsets prepared from PBMC isolated by either Ficoll or CPT protocol. a The mean number of CD19+, CD8+, CD14+, and CD4+ cells positively selected from Ficoll- and CPT-isolated PBMC collected from 6 healthy donors. The horizontal line denotes the means and each symbol represents an individual cell type isolated from Ficoll-PBMC (filled symbols) or CPT-PBMC (empty symbols) from each of 6 donors. b The mean viability of the same cell subsets. No significant differences ( P
Figure Legend Snippet: Yield and viability of immune cell subsets prepared from PBMC isolated by either Ficoll or CPT protocol. a The mean number of CD19+, CD8+, CD14+, and CD4+ cells positively selected from Ficoll- and CPT-isolated PBMC collected from 6 healthy donors. The horizontal line denotes the means and each symbol represents an individual cell type isolated from Ficoll-PBMC (filled symbols) or CPT-PBMC (empty symbols) from each of 6 donors. b The mean viability of the same cell subsets. No significant differences ( P

Techniques Used: Isolation, Cycling Probe Technology

Principle component analysis of gene expression showing distinct clusters between individual immune cell subsets but not between the same subsets derived from PBMC isolated by CPT or Ficoll protocols. Principle component analysis (PCA) was performed based on expression of all genes from the microarray. Individual immune cell types are represented by different colors: CD19+ B cells, blue; CD8+ T cells, green; CD14+ monocytes, purple; CD4+ T cells, orange; and total PBMC, red. The method used for PBMC isolation is represented by different symbols of differing size: CPT-based procedure by large circles and Ficoll-based procedure by small circles. PC#1, principle component #1; PC#2, principle component #2
Figure Legend Snippet: Principle component analysis of gene expression showing distinct clusters between individual immune cell subsets but not between the same subsets derived from PBMC isolated by CPT or Ficoll protocols. Principle component analysis (PCA) was performed based on expression of all genes from the microarray. Individual immune cell types are represented by different colors: CD19+ B cells, blue; CD8+ T cells, green; CD14+ monocytes, purple; CD4+ T cells, orange; and total PBMC, red. The method used for PBMC isolation is represented by different symbols of differing size: CPT-based procedure by large circles and Ficoll-based procedure by small circles. PC#1, principle component #1; PC#2, principle component #2

Techniques Used: Expressing, Derivative Assay, Isolation, Cycling Probe Technology, Microarray

Yield and quality of RNA recovered from total PBMC and their individual immune cell subsets did not significantly differ between PBMC isolated by Ficoll and CPT methods. a The mean amount of RNA per cell and ( b ) the mean RIN of RNA preparations extracted from total PBMC and their individual immune cell subsets derived from PBMC prepared by Ficoll and CPT methods from 6 health donors. There were no significant differences ( P
Figure Legend Snippet: Yield and quality of RNA recovered from total PBMC and their individual immune cell subsets did not significantly differ between PBMC isolated by Ficoll and CPT methods. a The mean amount of RNA per cell and ( b ) the mean RIN of RNA preparations extracted from total PBMC and their individual immune cell subsets derived from PBMC prepared by Ficoll and CPT methods from 6 health donors. There were no significant differences ( P

Techniques Used: Isolation, Cycling Probe Technology, Derivative Assay

Similar quantities of DNA with comparable quality were obtained from PBMC and immune cell subsets following either Ficoll or CPT protocol of PBMC isolation. a The mean amount of DNA extracted per cell and ( b ) the mean of the 260/280 ratios, as measure of DNA quality, were compared between DNA preparations obtained from total PBMC and immune cell subsets prepared from PBMC isolated by Ficoll and CPT methods from 6 healthy donors. There were no significant differences ( P
Figure Legend Snippet: Similar quantities of DNA with comparable quality were obtained from PBMC and immune cell subsets following either Ficoll or CPT protocol of PBMC isolation. a The mean amount of DNA extracted per cell and ( b ) the mean of the 260/280 ratios, as measure of DNA quality, were compared between DNA preparations obtained from total PBMC and immune cell subsets prepared from PBMC isolated by Ficoll and CPT methods from 6 healthy donors. There were no significant differences ( P

Techniques Used: Cycling Probe Technology, Isolation

Schematic outline of the study. PBMC from 6 healthy donors were isolated using Ficoll-Paque gradient fractionation or BD Vacutainer CPT protocol, and cell yield and purity were compared. Subsequently, immune cell subsets were separated by positive selection from PBMC obtained by both methods, and yields and viabilities of the subsets were determined and compared. RNA and DNA were extracted from total PBMC isolated by both protocols and from their subsets, and the nucleic acid yield and quality compared. Finally, gene expression analysis of individual immune cell subsets and total PBMC obtained by Ficoll and CPT isolation methods were performed and the results compared. LN 2 , liquid nitrogen
Figure Legend Snippet: Schematic outline of the study. PBMC from 6 healthy donors were isolated using Ficoll-Paque gradient fractionation or BD Vacutainer CPT protocol, and cell yield and purity were compared. Subsequently, immune cell subsets were separated by positive selection from PBMC obtained by both methods, and yields and viabilities of the subsets were determined and compared. RNA and DNA were extracted from total PBMC isolated by both protocols and from their subsets, and the nucleic acid yield and quality compared. Finally, gene expression analysis of individual immune cell subsets and total PBMC obtained by Ficoll and CPT isolation methods were performed and the results compared. LN 2 , liquid nitrogen

Techniques Used: Isolation, Fractionation, Cycling Probe Technology, Selection, Expressing

Yield and viability of PBMC isolated by the Ficoll and CPT protocols. a The mean numbers of PBMC per ml of blood obtained by Ficoll or CPT isolation procedure from 6 healthy donors. b The mean viability of PBMC freshly isolated from the same 6 healthy donors by either Ficoll gradient separation or CPT technique. No significant differences ( P
Figure Legend Snippet: Yield and viability of PBMC isolated by the Ficoll and CPT protocols. a The mean numbers of PBMC per ml of blood obtained by Ficoll or CPT isolation procedure from 6 healthy donors. b The mean viability of PBMC freshly isolated from the same 6 healthy donors by either Ficoll gradient separation or CPT technique. No significant differences ( P

Techniques Used: Isolation, Cycling Probe Technology

Purity of immune cell subsets obtained from PBMC isolated by either Ficoll or CPT procedures. CD19+, CD8+, CD14+, and CD4+ cell subsets were sequentially separated from PBMC isolated by Ficoll and CPT protocols from the same donor and their purity was determined by flow cytometry using antibodies against surface markers specific for individual immune cell types. Filled histograms were given by appropriate immunoglobulin isotype controls. Gates for determining positivity were established using isotype controls so that ~99.0 % of events were negative
Figure Legend Snippet: Purity of immune cell subsets obtained from PBMC isolated by either Ficoll or CPT procedures. CD19+, CD8+, CD14+, and CD4+ cell subsets were sequentially separated from PBMC isolated by Ficoll and CPT protocols from the same donor and their purity was determined by flow cytometry using antibodies against surface markers specific for individual immune cell types. Filled histograms were given by appropriate immunoglobulin isotype controls. Gates for determining positivity were established using isotype controls so that ~99.0 % of events were negative

Techniques Used: Isolation, Cycling Probe Technology, Flow Cytometry, Cytometry

Post-cryopreservation recovery and viability of PBMC isolated by the Ficoll and CPT methods. a The mean percent recovery after cryopreservation of PBMC collected using the Ficoll and CPT protocols. Percent recovery was calculated by dividing the number of PBMC recovered after thawing by the number of cells that were cryopreserved. b The mean viability of recovered PBMC isolated by the Ficoll and CPT technique. No significant differences ( P
Figure Legend Snippet: Post-cryopreservation recovery and viability of PBMC isolated by the Ficoll and CPT methods. a The mean percent recovery after cryopreservation of PBMC collected using the Ficoll and CPT protocols. Percent recovery was calculated by dividing the number of PBMC recovered after thawing by the number of cells that were cryopreserved. b The mean viability of recovered PBMC isolated by the Ficoll and CPT technique. No significant differences ( P

Techniques Used: Isolation, Cycling Probe Technology

30) Product Images from "The agr Inhibitors Solonamide B and Analogues Alter Immune Responses to Staphylococccus aureus but Do Not Exhibit Adverse Effects on Immune Cell Functions"

Article Title: The agr Inhibitors Solonamide B and Analogues Alter Immune Responses to Staphylococccus aureus but Do Not Exhibit Adverse Effects on Immune Cell Functions

Journal: PLoS ONE

doi: 10.1371/journal.pone.0145618

Solonamide-treated S . aureus influence cytokine production by immune cells. Bone marrow derived murine dendritic cells (A) and human peripheral blood mononuclear cells (B) were stimulated at an MOI of 10 with S . aureus strain 8325–4 pre-treated with SolB (2) ESB (4), Am16-L (9) or DMSO at 10 μg/mL. Protein concentrations of selected cytokines in the supernatants after 20–24 h incubation were measured by enzyme-linked immunosorbent assay for DCs and by cytometric bead array for human PBMCs. The results are based on 3 biological replicates and are normalized to the stimulus controls (LPS for DC; L . plantarum WCFS1 for PBMCs). Comparisons were made between the DMSO compound vehicle and the compounds.
Figure Legend Snippet: Solonamide-treated S . aureus influence cytokine production by immune cells. Bone marrow derived murine dendritic cells (A) and human peripheral blood mononuclear cells (B) were stimulated at an MOI of 10 with S . aureus strain 8325–4 pre-treated with SolB (2) ESB (4), Am16-L (9) or DMSO at 10 μg/mL. Protein concentrations of selected cytokines in the supernatants after 20–24 h incubation were measured by enzyme-linked immunosorbent assay for DCs and by cytometric bead array for human PBMCs. The results are based on 3 biological replicates and are normalized to the stimulus controls (LPS for DC; L . plantarum WCFS1 for PBMCs). Comparisons were made between the DMSO compound vehicle and the compounds.

Techniques Used: Derivative Assay, Incubation, Enzyme-linked Immunosorbent Assay

31) Product Images from "The Non-structural Protein 5 and Matrix Protein Are Antigenic Targets of T Cell Immunity to Genotype 1 Porcine Reproductive and Respiratory Syndrome Viruses"

Article Title: The Non-structural Protein 5 and Matrix Protein Are Antigenic Targets of T Cell Immunity to Genotype 1 Porcine Reproductive and Respiratory Syndrome Viruses

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2016.00040

Assessment of the phenotype and polyfunctionality of PRRSV-1 NSP5-specific CD8 T cells . Previously cryopreserved PBMC from identified T cell responder pigs on day 30 postinfection were stimulated with synthetic peptide pools representing M or NSP5 proteins or left unstimulated. The expression of CD44, CD62L, CD25, CD27, surface CD107a, and TNF-α by IFN-γ + CD4 T cells in response to M peptides and CD8 T cells in response to NSP5 peptides were assessed by flow cytometry as shown by representative dot plots. The mean % of unstimulated-corrected data from duplicate cultures are presented for individual animals and error bars show the SEM.
Figure Legend Snippet: Assessment of the phenotype and polyfunctionality of PRRSV-1 NSP5-specific CD8 T cells . Previously cryopreserved PBMC from identified T cell responder pigs on day 30 postinfection were stimulated with synthetic peptide pools representing M or NSP5 proteins or left unstimulated. The expression of CD44, CD62L, CD25, CD27, surface CD107a, and TNF-α by IFN-γ + CD4 T cells in response to M peptides and CD8 T cells in response to NSP5 peptides were assessed by flow cytometry as shown by representative dot plots. The mean % of unstimulated-corrected data from duplicate cultures are presented for individual animals and error bars show the SEM.

Techniques Used: Expressing, Flow Cytometry, Cytometry

Assessment of NSP5- and M-specific IFN–γ T cell responses over the course of infection and challenge with PRRSV-1 Olot/91 and SU1-Bel strains . Previously cryopreserved PBMC were stimulated ex vivo with synthetic peptide pools representing M or NSP5 protein or left unstimulated. IFN-γ expression by CD4 + CD8α low (CD4; open symbols) and CD4 − CD8α high (CD8; closed symbols) T cells from unstimulated (square symbols) or peptide pool-stimulated cultures (circle symbols) was assessed by flow cytometry. The mean % of IFN-γ + T cells from duplicate cultures are presented for each animal and error bars show the SEM. Data from peptide pool-stimulated cultures were compared against the corresponding unstimulated culture using a two-way ANOVA followed by a Tukey’s multiple comparison test.
Figure Legend Snippet: Assessment of NSP5- and M-specific IFN–γ T cell responses over the course of infection and challenge with PRRSV-1 Olot/91 and SU1-Bel strains . Previously cryopreserved PBMC were stimulated ex vivo with synthetic peptide pools representing M or NSP5 protein or left unstimulated. IFN-γ expression by CD4 + CD8α low (CD4; open symbols) and CD4 − CD8α high (CD8; closed symbols) T cells from unstimulated (square symbols) or peptide pool-stimulated cultures (circle symbols) was assessed by flow cytometry. The mean % of IFN-γ + T cells from duplicate cultures are presented for each animal and error bars show the SEM. Data from peptide pool-stimulated cultures were compared against the corresponding unstimulated culture using a two-way ANOVA followed by a Tukey’s multiple comparison test.

Techniques Used: Infection, Ex Vivo, Expressing, Flow Cytometry, Cytometry

Assessment of the conservation of identified T cell antigens M and NSP5 among PRRSV strains and assessment of T cell reactivity against variant peptides . The complete predicted amino acid sequences of M (from 64 PRRSV-1 and 31 PRRSV-2 strains) and NSP5 (from 19 PRRSV-1 and 36 PRRSV-2 strains) were aligned and the number of different amino acid variants at each residue plotted (A) . The predicted amino acid sequences of identified antigenic regions, M 29-43 , NSP5 13-27 , and NSP5 149-167 , were compared among the panel of 19 PRRSV-1 strains, for which both M and NSP5 sequence data were available, and a consensus sequence based on available PRRSV-2 strains (B) . Based on the observed amino acid substitutions, variant peptides were used to stimulate PBMC from pigs 71 and 86 (C) . IFN-γ expression by CD4 T cells to M 29-43 (pig 71) and CD8 T cells to NSP5 13-27 (pig 71), NSP5 145-159 (pig 86), and NSP5 153-170 (pig 71) peptides were assessed by flow cytometry. The mean% of unstimulated (medium) and peptide stimulated data from duplicate cultures are presented and error bars show the SEM. Values were compared to the unstimulated control using a one-way ANOVA followed by a Dunnett’s multiple comparison test; *** p
Figure Legend Snippet: Assessment of the conservation of identified T cell antigens M and NSP5 among PRRSV strains and assessment of T cell reactivity against variant peptides . The complete predicted amino acid sequences of M (from 64 PRRSV-1 and 31 PRRSV-2 strains) and NSP5 (from 19 PRRSV-1 and 36 PRRSV-2 strains) were aligned and the number of different amino acid variants at each residue plotted (A) . The predicted amino acid sequences of identified antigenic regions, M 29-43 , NSP5 13-27 , and NSP5 149-167 , were compared among the panel of 19 PRRSV-1 strains, for which both M and NSP5 sequence data were available, and a consensus sequence based on available PRRSV-2 strains (B) . Based on the observed amino acid substitutions, variant peptides were used to stimulate PBMC from pigs 71 and 86 (C) . IFN-γ expression by CD4 T cells to M 29-43 (pig 71) and CD8 T cells to NSP5 13-27 (pig 71), NSP5 145-159 (pig 86), and NSP5 153-170 (pig 71) peptides were assessed by flow cytometry. The mean% of unstimulated (medium) and peptide stimulated data from duplicate cultures are presented and error bars show the SEM. Values were compared to the unstimulated control using a one-way ANOVA followed by a Dunnett’s multiple comparison test; *** p

Techniques Used: Variant Assay, Sequencing, Expressing, Flow Cytometry, Cytometry

32) Product Images from "Immune and Viral Correlates of "Secondary Viral Control" after Treatment Interruption in Chronically HIV-1 Infected Patients"

Article Title: Immune and Viral Correlates of "Secondary Viral Control" after Treatment Interruption in Chronically HIV-1 Infected Patients

Journal: PLoS ONE

doi: 10.1371/journal.pone.0037792

Evaluation of T cell polyfunctionality. PBMC from all study subjects were stimulated with peptide pools, selected for positive responses in the IFN-γ ELISPOT screening in each individual separately. In A the gating strategy for identification of multifunctional CD3+CD8+ and CD3+CD8− (CD4+) T cell responses is shown. After physical gating the mononuclear cells and excluding the dead cells, CD3+CD8+T-cells and CD3+CD8−(CD4+ T-cells) were identified. Within each of these populations the expression of IFN-γ, IL-2, TNF-a or CD107a was plotted against side scatter, to allow Boolean gating for the ultimate quantification of polyfunctionality. Polyfunctionality was analyzed using Flowjo by assessing the percentages of CD3+CD8− (hence CD4+) T cells (in B) and CD3+ CD8+ T cells (in C) that produce one, two, three or four cytokines. Percentages of co-expression for all selected peptide pools within one patient were summed and the mean+SD was calculated for each group. In D and E is the number of CD4+ T-cells respectively CD8+ T-cells that produce one, two, three or four cytokines presented. No statistical differences between groups were found.
Figure Legend Snippet: Evaluation of T cell polyfunctionality. PBMC from all study subjects were stimulated with peptide pools, selected for positive responses in the IFN-γ ELISPOT screening in each individual separately. In A the gating strategy for identification of multifunctional CD3+CD8+ and CD3+CD8− (CD4+) T cell responses is shown. After physical gating the mononuclear cells and excluding the dead cells, CD3+CD8+T-cells and CD3+CD8−(CD4+ T-cells) were identified. Within each of these populations the expression of IFN-γ, IL-2, TNF-a or CD107a was plotted against side scatter, to allow Boolean gating for the ultimate quantification of polyfunctionality. Polyfunctionality was analyzed using Flowjo by assessing the percentages of CD3+CD8− (hence CD4+) T cells (in B) and CD3+ CD8+ T cells (in C) that produce one, two, three or four cytokines. Percentages of co-expression for all selected peptide pools within one patient were summed and the mean+SD was calculated for each group. In D and E is the number of CD4+ T-cells respectively CD8+ T-cells that produce one, two, three or four cytokines presented. No statistical differences between groups were found.

Techniques Used: Enzyme-linked Immunospot, Expressing

33) Product Images from "Clinical Significance of IL-23 Regulating IL-17A and/or IL-17F Positive Th17 Cells in Chronic Periodontitis"

Article Title: Clinical Significance of IL-23 Regulating IL-17A and/or IL-17F Positive Th17 Cells in Chronic Periodontitis

Journal: Mediators of Inflammation

doi: 10.1155/2014/627959

Purity verification of separated CD4 + T cells by flow cytometry. (a) showed the isotype control performed by Simultest IgG2a/IgG1. (b) represents double staining by anti-human CD3 and CD4 antibody. (c) represents anti-human antibody CD4 staining. These results showed that the purity of CD4 + from PBMC can reach a percent of more than 95%.
Figure Legend Snippet: Purity verification of separated CD4 + T cells by flow cytometry. (a) showed the isotype control performed by Simultest IgG2a/IgG1. (b) represents double staining by anti-human CD3 and CD4 antibody. (c) represents anti-human antibody CD4 staining. These results showed that the purity of CD4 + from PBMC can reach a percent of more than 95%.

Techniques Used: Flow Cytometry, Cytometry, Double Staining, Staining

34) Product Images from "Early Establishment and Antigen Dependence of Simian Immunodeficiency Virus-Specific CD8+ T-Cell Defects ▿"

Article Title: Early Establishment and Antigen Dependence of Simian Immunodeficiency Virus-Specific CD8+ T-Cell Defects ▿

Journal: Journal of Virology

doi: 10.1128/JVI.00813-07

SIV-specific CD8 + T cells are highly sensitive to apoptosis, which is inhibited by IL-15. PBMC from uninfected and SIV-infected rhesus macaques were cultured in the presence or absence of sFasL (10 ng/ml) overnight, and spontaneous and CD95-induced apoptosis was analyzed. Gag-specific CD8 + T cells from SIV-infected animals were identified using the Gag CM9 tetramer. (A) Pooled data showing the percentages of spontaneous and CD95-induced apoptosis for CD8 + T cells from uninfected rhesus macaques ( n = 18) and for total ( n = 15) and Gag-specific ( n = 15) CD8 + T cells from SIV-infected rhesus macaques in overnight cultures of PBMC. Horizontal lines indicate means. (B) Pooled data showing the percentages of spontaneous and CD95-induced apoptosis in the presence and absence of 5 ng/ml IL-15 for CD8 + T cells from uninfected rhesus macaques ( n = 18) and for total ( n = 15) and Gag-specific ( n = 15) CD8 + T cells from SIV-infected rhesus macaques in overnight cultures of PBMC. Horizontal lines indicate means. (C) CD95-specific apoptosis of total CD8 + T cells from uninfected rhesus macaques and of total and Gag-specific CD8 + T cells from SIV-infected rhesus macaques. For calculation of specific apoptosis, see Materials and Methods. Horizontal lines indicate means. Lines with asterisks at the top indicate statistical significance. P values are given in the key. (D) Pooled data showing the percentages of spontaneous and CD95-induced apoptosis of naïve and memory CD8 + T-cell subpopulations of chronically SIV infected rhesus macaques ( n = 5). (E) (Left) Pooled data showing MFI of Bcl-2 expression in total CD8 + T cells from 16 uninfected macaques and in Gag-specific and total CD8 + T cells from 15 SIV-infected rhesus macaques. (Right) Bcl-2 MFIs after 14 h in culture in the presence or absence of IL-15 (5 ng/ml) for total CD8 + T cells from uninfected controls ( n = 6) and for total ( n = 7) and Gag-specific ( n = 7) CD8 + T cells from SIV-infected animals. (F) (Left) MFI of Bcl-x L expression in total CD8 + T cells from 16 uninfected macaques and in Gag-specific and total CD8 + T cells from 15 SIV-infected rhesus macaques. (Right) Bcl-x L MFIs after 14 h in culture in the presence or absence of IL-15 (5 ng/ml) for total CD8 + T cells from uninfected controls ( n = 6) and total ( n = 7) and Gag-specific ( n = 7) CD8 + T cells from SIV-infected animals.
Figure Legend Snippet: SIV-specific CD8 + T cells are highly sensitive to apoptosis, which is inhibited by IL-15. PBMC from uninfected and SIV-infected rhesus macaques were cultured in the presence or absence of sFasL (10 ng/ml) overnight, and spontaneous and CD95-induced apoptosis was analyzed. Gag-specific CD8 + T cells from SIV-infected animals were identified using the Gag CM9 tetramer. (A) Pooled data showing the percentages of spontaneous and CD95-induced apoptosis for CD8 + T cells from uninfected rhesus macaques ( n = 18) and for total ( n = 15) and Gag-specific ( n = 15) CD8 + T cells from SIV-infected rhesus macaques in overnight cultures of PBMC. Horizontal lines indicate means. (B) Pooled data showing the percentages of spontaneous and CD95-induced apoptosis in the presence and absence of 5 ng/ml IL-15 for CD8 + T cells from uninfected rhesus macaques ( n = 18) and for total ( n = 15) and Gag-specific ( n = 15) CD8 + T cells from SIV-infected rhesus macaques in overnight cultures of PBMC. Horizontal lines indicate means. (C) CD95-specific apoptosis of total CD8 + T cells from uninfected rhesus macaques and of total and Gag-specific CD8 + T cells from SIV-infected rhesus macaques. For calculation of specific apoptosis, see Materials and Methods. Horizontal lines indicate means. Lines with asterisks at the top indicate statistical significance. P values are given in the key. (D) Pooled data showing the percentages of spontaneous and CD95-induced apoptosis of naïve and memory CD8 + T-cell subpopulations of chronically SIV infected rhesus macaques ( n = 5). (E) (Left) Pooled data showing MFI of Bcl-2 expression in total CD8 + T cells from 16 uninfected macaques and in Gag-specific and total CD8 + T cells from 15 SIV-infected rhesus macaques. (Right) Bcl-2 MFIs after 14 h in culture in the presence or absence of IL-15 (5 ng/ml) for total CD8 + T cells from uninfected controls ( n = 6) and for total ( n = 7) and Gag-specific ( n = 7) CD8 + T cells from SIV-infected animals. (F) (Left) MFI of Bcl-x L expression in total CD8 + T cells from 16 uninfected macaques and in Gag-specific and total CD8 + T cells from 15 SIV-infected rhesus macaques. (Right) Bcl-x L MFIs after 14 h in culture in the presence or absence of IL-15 (5 ng/ml) for total CD8 + T cells from uninfected controls ( n = 6) and total ( n = 7) and Gag-specific ( n = 7) CD8 + T cells from SIV-infected animals.

Techniques Used: Infection, Cell Culture, Expressing

35) Product Images from "Distinct susceptibility of HIV vaccine vector-induced CD4 T cells to HIV infection"

Article Title: Distinct susceptibility of HIV vaccine vector-induced CD4 T cells to HIV infection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006888

ALVAC-specific CD4 T cells are markedly less susceptible to HIV infection in vitro than Ad5 vector-specific CD4 T cells. PBMC collected from ALVAC- (RV144) or Ad5-vectored (HVTN204) HIV vaccine recipients were stained with CFSE and then re-stimulated with the recall vector antigen (ALVAC or Ad5) for three days before being infected with CCR5-tropic (US-1 strain) ( A ) or CXCR4-tropic (92/UG/029 strain) ( B ) HIV. HIV infection rate in vector-specific CD4 T cells was determined using flow cytometry to measure p24 expression 3 days post infection and expressed as the percentage of p24 + CFSE-low CD4 T cells. Representative flow cytometry plots shown at left are gated on CD3 + CD8 - CD4 T cells. Statistical analysis was performed using an unpaired Student’s t test. *p ≤ 0.05, **p ≤ 0.01.
Figure Legend Snippet: ALVAC-specific CD4 T cells are markedly less susceptible to HIV infection in vitro than Ad5 vector-specific CD4 T cells. PBMC collected from ALVAC- (RV144) or Ad5-vectored (HVTN204) HIV vaccine recipients were stained with CFSE and then re-stimulated with the recall vector antigen (ALVAC or Ad5) for three days before being infected with CCR5-tropic (US-1 strain) ( A ) or CXCR4-tropic (92/UG/029 strain) ( B ) HIV. HIV infection rate in vector-specific CD4 T cells was determined using flow cytometry to measure p24 expression 3 days post infection and expressed as the percentage of p24 + CFSE-low CD4 T cells. Representative flow cytometry plots shown at left are gated on CD3 + CD8 - CD4 T cells. Statistical analysis was performed using an unpaired Student’s t test. *p ≤ 0.05, **p ≤ 0.01.

Techniques Used: Infection, In Vitro, Plasmid Preparation, Staining, Flow Cytometry, Cytometry, Expressing

ALVAC vector-specific CD4 T cells express lower levels of the HIV co-receptors CCR5 and CXCR4 than Ad5 vector-specific CD4 T cells. PBMC of RV144 and HVTN204 vaccine recipients were stained with CFSE and stimulated with vector (ALVAC or Ad5) for 6 days. Surface expression of CCR5 ( A ) and CXCR4 ( B ) was measured by flow cytometry. Representative flow cytometry dot plots (left; gated on CD3+CD8- CD4 T cells) and histogram for co-receptor expression on ALVAC- and Ad5 vector-specific CD4 T cells are shown. Comparison of % CCR5+ or CXCR4+ vector-specific CD4 T cells from multiple subjects is shown (right). (C) HIV infection in co-receptor+ vs. co-receptor-, Ad5-specific CD4 T cells. CFSE-low, Ad5-specific CD4 T cells were gated for analysis (left). HIV infection rate (% p24+) in CCR5+ vs. CCR5- Ad5-specific CD4 T cells infected with R5 HIV (middle) or in CXCR4+ vs CXCR4- Ad5-specific CD4 T cells infected with X4 HIV (right) were shown. For both R5 and X4, no HIV infection was included as control to set p24 staining gate. Statistical analysis was performed using an unpaired Student’s t test; *p ≤ 0.05, **p ≤ 0.01.
Figure Legend Snippet: ALVAC vector-specific CD4 T cells express lower levels of the HIV co-receptors CCR5 and CXCR4 than Ad5 vector-specific CD4 T cells. PBMC of RV144 and HVTN204 vaccine recipients were stained with CFSE and stimulated with vector (ALVAC or Ad5) for 6 days. Surface expression of CCR5 ( A ) and CXCR4 ( B ) was measured by flow cytometry. Representative flow cytometry dot plots (left; gated on CD3+CD8- CD4 T cells) and histogram for co-receptor expression on ALVAC- and Ad5 vector-specific CD4 T cells are shown. Comparison of % CCR5+ or CXCR4+ vector-specific CD4 T cells from multiple subjects is shown (right). (C) HIV infection in co-receptor+ vs. co-receptor-, Ad5-specific CD4 T cells. CFSE-low, Ad5-specific CD4 T cells were gated for analysis (left). HIV infection rate (% p24+) in CCR5+ vs. CCR5- Ad5-specific CD4 T cells infected with R5 HIV (middle) or in CXCR4+ vs CXCR4- Ad5-specific CD4 T cells infected with X4 HIV (right) were shown. For both R5 and X4, no HIV infection was included as control to set p24 staining gate. Statistical analysis was performed using an unpaired Student’s t test; *p ≤ 0.05, **p ≤ 0.01.

Techniques Used: Plasmid Preparation, Staining, Expressing, Flow Cytometry, Cytometry, Infection

ALVAC- and Ad5-specific CD4 T cells show similar levels of innate antiviral gene expression and immune activation. (A) Relative expression of innate antiviral genes in ALVAC- and Ad5-specific CD4 T cells. RV144 and HVTN204 PBMC were CFSE-labeled and vector stimulated as described above. On day 6 ALVAC- and Ad5-specific CD4 T cells were sorted from PBMC based on CFSE-low and subjected to quantitative PCR for analysis of gene expression. The results were shown as fold change of ALVAC relative to Ad5. (B) HIV infection of ALVAC-specific CD4 T cells in RV144 PBMC in the presence or absence of anti-human IFNAR antibody blockade (gated on CD3+CD8- CD4 T cells). Number in each plot shows %p24+ in CFSE-low CD4 T cells. (C) Surface expression of T-cell activation markers CD25 (top) and CD69 (bottom) on ALVAC- vs. Ad5-specific CD4 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8- CD4 T cells). Number in each plot shows % CD25+ or % CD69+ in CFSE-low CD4 T cells.
Figure Legend Snippet: ALVAC- and Ad5-specific CD4 T cells show similar levels of innate antiviral gene expression and immune activation. (A) Relative expression of innate antiviral genes in ALVAC- and Ad5-specific CD4 T cells. RV144 and HVTN204 PBMC were CFSE-labeled and vector stimulated as described above. On day 6 ALVAC- and Ad5-specific CD4 T cells were sorted from PBMC based on CFSE-low and subjected to quantitative PCR for analysis of gene expression. The results were shown as fold change of ALVAC relative to Ad5. (B) HIV infection of ALVAC-specific CD4 T cells in RV144 PBMC in the presence or absence of anti-human IFNAR antibody blockade (gated on CD3+CD8- CD4 T cells). Number in each plot shows %p24+ in CFSE-low CD4 T cells. (C) Surface expression of T-cell activation markers CD25 (top) and CD69 (bottom) on ALVAC- vs. Ad5-specific CD4 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8- CD4 T cells). Number in each plot shows % CD25+ or % CD69+ in CFSE-low CD4 T cells.

Techniques Used: Expressing, Activation Assay, Labeling, Plasmid Preparation, Real-time Polymerase Chain Reaction, Infection

ALVAC elicits distinct profile of vector-specific CD8 vs. CD4 T-cell proliferative response compared to Ad5 vector. PBMC were stained with CFSE and re-stimulated with the corresponding vector for 6 days. (A) Representative flow cytometry plots for PBMC of multiple subjects showing vector-induced CD8 vs. CD4 T-cell proliferative responses in PBMC of RV144 (top) and HVTN204 (bottom) vaccine recipients. (B) Comparison for vector-specific CD8 and CD4 T-cell proliferative responses (% CFSE-low) in PBMC of RV144 and HVTN204 after corresponding vector stimulation. (C) Ratio of vector-specific CD8/CD4 T-cell proliferation in RV144 (ALVAC) and HVTN204 (Ad5) PBMC. Statistical analysis was performed using an unpaired Student’s t test; n = 14. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Figure Legend Snippet: ALVAC elicits distinct profile of vector-specific CD8 vs. CD4 T-cell proliferative response compared to Ad5 vector. PBMC were stained with CFSE and re-stimulated with the corresponding vector for 6 days. (A) Representative flow cytometry plots for PBMC of multiple subjects showing vector-induced CD8 vs. CD4 T-cell proliferative responses in PBMC of RV144 (top) and HVTN204 (bottom) vaccine recipients. (B) Comparison for vector-specific CD8 and CD4 T-cell proliferative responses (% CFSE-low) in PBMC of RV144 and HVTN204 after corresponding vector stimulation. (C) Ratio of vector-specific CD8/CD4 T-cell proliferation in RV144 (ALVAC) and HVTN204 (Ad5) PBMC. Statistical analysis was performed using an unpaired Student’s t test; n = 14. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

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

ALVAC-induced CD8 T cells manifest stronger antiviral and cytotoxic phenotype than Ad5 vector-induced CD8 T cells. PBMC of vaccine recipients were stained with CFSE and then stimulated with vector antigen for 6 days, followed by brief PMA/Ionomycin re-stimulation (6 hours) for cytokine/effector molecule re-synthesis. Intracellular staining and flow cytometry were used to measure the production of IFN-γ, MIP-1β, perforin, granzyme B (GZMB), and CD107a; results are expressed as % cytokine+ in CFSE-low CD8 T cells. Statistical analysis was performed using an unpaired Student’s t test; n = 3–5. n.s.: not significant; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Figure Legend Snippet: ALVAC-induced CD8 T cells manifest stronger antiviral and cytotoxic phenotype than Ad5 vector-induced CD8 T cells. PBMC of vaccine recipients were stained with CFSE and then stimulated with vector antigen for 6 days, followed by brief PMA/Ionomycin re-stimulation (6 hours) for cytokine/effector molecule re-synthesis. Intracellular staining and flow cytometry were used to measure the production of IFN-γ, MIP-1β, perforin, granzyme B (GZMB), and CD107a; results are expressed as % cytokine+ in CFSE-low CD8 T cells. Statistical analysis was performed using an unpaired Student’s t test; n = 3–5. n.s.: not significant; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

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

Phenotypic characterization of ALVAC- and Ad5 vector-specific CD4 T cells. PBMC from RV144 or HVTN204 vaccine recipients were stained with CFSE and re-stimulated with vector for 6 days. Phenotypes and cytokine profile of CFSE-low, vector-specific CD4 T cells were measured by flow cytometry. (A) Comparison for percent of central memory (CM: CCR7+CD45RO+) and effector memory (EM: CCR7-CD45R)+) subsets in CFSE-low, ALVAC- and Ad5 vector-specific CD4 T cells; (B) Comparison for α4β7+% in CFSE-low, ALVAC- and Ad5 vector-specific CD4 T cells; (C) Representative flow cytometric plots for cytokine expression (IFN-γ, IL-2, and IL-17) in CFSE-low, ALVAC-specific (top) or Ad5 vector-specific (bottom) CD4 T cells; (D) Comparison for cytokine expression in CFSE-low, vector-specific CD4 T cells (% cytokine+ CFSE-low) between ALVAC and Ad5 vector from multiple vaccine recipients (n = 11). n.s.: not significant, *p ≤ 0.05, **p ≤ 0.01.
Figure Legend Snippet: Phenotypic characterization of ALVAC- and Ad5 vector-specific CD4 T cells. PBMC from RV144 or HVTN204 vaccine recipients were stained with CFSE and re-stimulated with vector for 6 days. Phenotypes and cytokine profile of CFSE-low, vector-specific CD4 T cells were measured by flow cytometry. (A) Comparison for percent of central memory (CM: CCR7+CD45RO+) and effector memory (EM: CCR7-CD45R)+) subsets in CFSE-low, ALVAC- and Ad5 vector-specific CD4 T cells; (B) Comparison for α4β7+% in CFSE-low, ALVAC- and Ad5 vector-specific CD4 T cells; (C) Representative flow cytometric plots for cytokine expression (IFN-γ, IL-2, and IL-17) in CFSE-low, ALVAC-specific (top) or Ad5 vector-specific (bottom) CD4 T cells; (D) Comparison for cytokine expression in CFSE-low, vector-specific CD4 T cells (% cytokine+ CFSE-low) between ALVAC and Ad5 vector from multiple vaccine recipients (n = 11). n.s.: not significant, *p ≤ 0.05, **p ≤ 0.01.

Techniques Used: Plasmid Preparation, Staining, Flow Cytometry, Cytometry, Expressing

CD8 depletion increases HIV susceptibility of ALVAC-specific CD4 T cells. (A) Representative flow cytometry plots showing HIV infection in CFSE-low, vector-specific CD4 T cells in whole (CD8+) or CD8-depleted (CD8-) PBMC. Whole and CD8-depleted PBMC were CFSE stained and stimulated with vector antigen for 3 days before being infected with CCR5- or CXCR4-tropic HIV. HIV infection rate was determined using flow cytometry to measure intracellular p24 and expressed as the percentage of p24+ in CFSE-low CD4 T cells. CD3+CD8- T cells were gated for analysis. (B) Comparison for HIV infection rates in CFSE-low vector-specific CD4 T cells (% p24+) in whole or CD8-depleted PBMC from multiple vaccine recipients. (C) HIV infection (% p24+) in ALVAC-specific CD4 T cells in whole PBMC, CD8-depleted PBMC or PBMC from which CD8 T cells have been depleted and then added back to culture in trans-well (gated on CD3+CD8- CD4 T cells). % p24+ in CFSE-low cells was shown. (D) MIP-1β expression in ALVAC- versus Ad5-specific CD8 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8+ T cells). (E) Flow cytometry plot (left) and bar graph (right) showing the viability of ALVAC-specific CD4 T cells (based on Aqua Blue staining) 6 days after vector stimulation with or without CD8 T-cell depletion (gated on CD3+CD8- CD4 T cells). Statistical analysis was performed using an unpaired Student’s t test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Figure Legend Snippet: CD8 depletion increases HIV susceptibility of ALVAC-specific CD4 T cells. (A) Representative flow cytometry plots showing HIV infection in CFSE-low, vector-specific CD4 T cells in whole (CD8+) or CD8-depleted (CD8-) PBMC. Whole and CD8-depleted PBMC were CFSE stained and stimulated with vector antigen for 3 days before being infected with CCR5- or CXCR4-tropic HIV. HIV infection rate was determined using flow cytometry to measure intracellular p24 and expressed as the percentage of p24+ in CFSE-low CD4 T cells. CD3+CD8- T cells were gated for analysis. (B) Comparison for HIV infection rates in CFSE-low vector-specific CD4 T cells (% p24+) in whole or CD8-depleted PBMC from multiple vaccine recipients. (C) HIV infection (% p24+) in ALVAC-specific CD4 T cells in whole PBMC, CD8-depleted PBMC or PBMC from which CD8 T cells have been depleted and then added back to culture in trans-well (gated on CD3+CD8- CD4 T cells). % p24+ in CFSE-low cells was shown. (D) MIP-1β expression in ALVAC- versus Ad5-specific CD8 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8+ T cells). (E) Flow cytometry plot (left) and bar graph (right) showing the viability of ALVAC-specific CD4 T cells (based on Aqua Blue staining) 6 days after vector stimulation with or without CD8 T-cell depletion (gated on CD3+CD8- CD4 T cells). Statistical analysis was performed using an unpaired Student’s t test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Techniques Used: Flow Cytometry, Cytometry, Infection, Plasmid Preparation, Staining, Expressing

ALVAC-specific CD4 T cells produce higher levels of MIP-1β than Ad5-specific CD4 T cells, which contributes partly to their lower susceptibility to in vitro HIV infection. (A) MIP-1β expression in CFSE-low, vector-specific CD4 T cells was determined by intracellular cytokine staining and flow cytometric analysis as described above; results are expressed as % MIP-1β+ CFSE-low CD4 T cells (n = 11). (B) Impact of MIP-1β neutralization on HIV infection of ALVAC-specific CD4 T cells. PBMC were stained with CFSE and re-stimulated in vitro with ALVAC vector in the absence of presence of β-chemokine neutralizing antibodies (CCL3/4/5). 3 days after vector stimulation, PBMC were infected with R5 HIV, followed by measurement of HIV infection in vector-specific CD4 T cells (CFSE-low, CD4 T cells) on day 6 after initial vector stimulation. HIV infection was expressed as the percentage of p24+ in CFSE-low CD4 T cells (n = 4). *p ≤ 0.05, **p ≤ 0.01.
Figure Legend Snippet: ALVAC-specific CD4 T cells produce higher levels of MIP-1β than Ad5-specific CD4 T cells, which contributes partly to their lower susceptibility to in vitro HIV infection. (A) MIP-1β expression in CFSE-low, vector-specific CD4 T cells was determined by intracellular cytokine staining and flow cytometric analysis as described above; results are expressed as % MIP-1β+ CFSE-low CD4 T cells (n = 11). (B) Impact of MIP-1β neutralization on HIV infection of ALVAC-specific CD4 T cells. PBMC were stained with CFSE and re-stimulated in vitro with ALVAC vector in the absence of presence of β-chemokine neutralizing antibodies (CCL3/4/5). 3 days after vector stimulation, PBMC were infected with R5 HIV, followed by measurement of HIV infection in vector-specific CD4 T cells (CFSE-low, CD4 T cells) on day 6 after initial vector stimulation. HIV infection was expressed as the percentage of p24+ in CFSE-low CD4 T cells (n = 4). *p ≤ 0.05, **p ≤ 0.01.

Techniques Used: In Vitro, Infection, Expressing, Plasmid Preparation, Staining, Flow Cytometry, Neutralization

ALVAC-induced CD8 T cells inhibit the expansion of autologous vector-specific CD4 T cells. (A) CD8+ cells were depleted from PBMC of vaccine recipients using magnetic beads. CD8-depleted or whole PBMC were CFSE stained and re-stimulated with the appropriate vector for 6 days. Efficient CD8 depletion was verified by flow cytometry. Number in the bottom-left quadrant shows % CFSE-low, proliferating CD4 T cells in total CD4 T cells. (B) Comparison for vector-specific CD4 T cell proliferation (% CFSE-low) in PBMC with or without CD8 depletion (n = 7 for ALVAC; n = 4 for Ad5). (C) CD4 T-cell proliferation in RV144 PBMC 6 days after stimulation with ALVAC. Comparison of whole PBMC, CD8-depleted PBMC, and PBMC from which CD8 T cells were depleted and then added back to culture in trans-well (gated on CD3+ T cells). (D) CD25 and FoxP3 expression in ALVAC- versus Ad5-specific CD8 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8+ CFSE-low T cells). (E) Flow cytometry plot and (F) bar graph showing CD4 T cell viability (% viable cells) in RV144 PBMC 3 days after stimulation with ALVAC (before significant T-cell proliferation occurs), as determined by Aqua Blue dye exclusion. Comparison of cell viability in whole PBMC, CD8-depleted PBMC, and PBMC from which CD8 T cells were depleted then added back to culture in trans-well. Statistical analysis was performed using an unpaired Student’s t test; n = 2 (Ad5) or 7 (ALVAC). n.s.: non-significant; *p ≤ 0.05, **p ≤ 0.01.
Figure Legend Snippet: ALVAC-induced CD8 T cells inhibit the expansion of autologous vector-specific CD4 T cells. (A) CD8+ cells were depleted from PBMC of vaccine recipients using magnetic beads. CD8-depleted or whole PBMC were CFSE stained and re-stimulated with the appropriate vector for 6 days. Efficient CD8 depletion was verified by flow cytometry. Number in the bottom-left quadrant shows % CFSE-low, proliferating CD4 T cells in total CD4 T cells. (B) Comparison for vector-specific CD4 T cell proliferation (% CFSE-low) in PBMC with or without CD8 depletion (n = 7 for ALVAC; n = 4 for Ad5). (C) CD4 T-cell proliferation in RV144 PBMC 6 days after stimulation with ALVAC. Comparison of whole PBMC, CD8-depleted PBMC, and PBMC from which CD8 T cells were depleted and then added back to culture in trans-well (gated on CD3+ T cells). (D) CD25 and FoxP3 expression in ALVAC- versus Ad5-specific CD8 T cells 6 days after stimulation with the corresponding vector (gated on CD3+CD8+ CFSE-low T cells). (E) Flow cytometry plot and (F) bar graph showing CD4 T cell viability (% viable cells) in RV144 PBMC 3 days after stimulation with ALVAC (before significant T-cell proliferation occurs), as determined by Aqua Blue dye exclusion. Comparison of cell viability in whole PBMC, CD8-depleted PBMC, and PBMC from which CD8 T cells were depleted then added back to culture in trans-well. Statistical analysis was performed using an unpaired Student’s t test; n = 2 (Ad5) or 7 (ALVAC). n.s.: non-significant; *p ≤ 0.05, **p ≤ 0.01.

Techniques Used: Plasmid Preparation, Magnetic Beads, Staining, Flow Cytometry, Cytometry, Expressing

36) Product Images from "Analyses of the peripheral immunome following multiple administrations of avelumab, a human IgG1 anti-PD-L1 monoclonal antibody"

Article Title: Analyses of the peripheral immunome following multiple administrations of avelumab, a human IgG1 anti-PD-L1 monoclonal antibody

Journal: Journal for Immunotherapy of Cancer

doi: 10.1186/s40425-017-0220-y

ADCC assay using PBMC from healthy donors or H441 human lung tumor cells as targets. a PD-L1 expression in H441 cells. b NK cells were purified from PBMC from five healthy donors using negative magnetic selection. In vitro ADCC assays were performed at effector:target ratios of 25:1, 12.5:1, and 6.25:1, using an IgG1 isotype control antibody (gray bars, 1 ng/mL) or avelumab (black bars, 1 ng/mL). Results are displayed as mean + SEM of triplicate wells
Figure Legend Snippet: ADCC assay using PBMC from healthy donors or H441 human lung tumor cells as targets. a PD-L1 expression in H441 cells. b NK cells were purified from PBMC from five healthy donors using negative magnetic selection. In vitro ADCC assays were performed at effector:target ratios of 25:1, 12.5:1, and 6.25:1, using an IgG1 isotype control antibody (gray bars, 1 ng/mL) or avelumab (black bars, 1 ng/mL). Results are displayed as mean + SEM of triplicate wells

Techniques Used: ADCC Assay, Expressing, Purification, Selection, In Vitro

ADCC assay using NK cells from a cancer patient with NSCLC against autologous PBMC sorted to enrich for PD-L1, or H460 or H441 human lung tumor cells. a PD-L1 expression in total PBMC, and PBMC magnetically sorted via negative and positive selection to enrich for PD-L1 negative and PD-L1 positive fractions, respectively. b PD-L1 expression in H460 and H441 cells. c NK cells were purified from PBMC from a cancer patient using negative magnetic selection. In vitro ADCC assays were performed at effector:target ratio of 25:1, using an IgG1 isotype control antibody (gray bars, 1 ng/mL) or avelumab (black bars, 1 ng/mL). Results are displayed as mean + SEM of triplicate wells. Data analyzed with unpaired T -test comparing avelumab vs isotype control; ** p
Figure Legend Snippet: ADCC assay using NK cells from a cancer patient with NSCLC against autologous PBMC sorted to enrich for PD-L1, or H460 or H441 human lung tumor cells. a PD-L1 expression in total PBMC, and PBMC magnetically sorted via negative and positive selection to enrich for PD-L1 negative and PD-L1 positive fractions, respectively. b PD-L1 expression in H460 and H441 cells. c NK cells were purified from PBMC from a cancer patient using negative magnetic selection. In vitro ADCC assays were performed at effector:target ratio of 25:1, using an IgG1 isotype control antibody (gray bars, 1 ng/mL) or avelumab (black bars, 1 ng/mL). Results are displayed as mean + SEM of triplicate wells. Data analyzed with unpaired T -test comparing avelumab vs isotype control; ** p

Techniques Used: ADCC Assay, Expressing, Selection, Purification, In Vitro

ADCC assay using PBMC from healthy donors or H441 human lung tumor cells as targets. a PD-L1 expression in H441 cells. b NK cells were purified from PBMC from five healthy donors using negative magnetic selection. In vitro ADCC assays were performed at effector:target ratios of 25:1, 12.5:1, and 6.25:1, using an IgG1 isotype control antibody (gray bars, 1 ng/mL) or avelumab (black bars, 1 ng/mL). Results are displayed as mean + SEM of triplicate wells
Figure Legend Snippet: ADCC assay using PBMC from healthy donors or H441 human lung tumor cells as targets. a PD-L1 expression in H441 cells. b NK cells were purified from PBMC from five healthy donors using negative magnetic selection. In vitro ADCC assays were performed at effector:target ratios of 25:1, 12.5:1, and 6.25:1, using an IgG1 isotype control antibody (gray bars, 1 ng/mL) or avelumab (black bars, 1 ng/mL). Results are displayed as mean + SEM of triplicate wells

Techniques Used: ADCC Assay, Expressing, Purification, Selection, In Vitro

ADCC assay using NK cells from a cancer patient with NSCLC against autologous PBMC sorted to enrich for PD-L1, or H460 or H441 human lung tumor cells. a PD-L1 expression in total PBMC, and PBMC magnetically sorted via negative and positive selection to enrich for PD-L1 negative and PD-L1 positive fractions, respectively. b PD-L1 expression in H460 and H441 cells. c NK cells were purified from PBMC from a cancer patient using negative magnetic selection. In vitro ADCC assays were performed at effector:target ratio of 25:1, using an IgG1 isotype control antibody (gray bars, 1 ng/mL) or avelumab (black bars, 1 ng/mL). Results are displayed as mean + SEM of triplicate wells. Data analyzed with unpaired T -test comparing avelumab vs isotype control; ** p
Figure Legend Snippet: ADCC assay using NK cells from a cancer patient with NSCLC against autologous PBMC sorted to enrich for PD-L1, or H460 or H441 human lung tumor cells. a PD-L1 expression in total PBMC, and PBMC magnetically sorted via negative and positive selection to enrich for PD-L1 negative and PD-L1 positive fractions, respectively. b PD-L1 expression in H460 and H441 cells. c NK cells were purified from PBMC from a cancer patient using negative magnetic selection. In vitro ADCC assays were performed at effector:target ratio of 25:1, using an IgG1 isotype control antibody (gray bars, 1 ng/mL) or avelumab (black bars, 1 ng/mL). Results are displayed as mean + SEM of triplicate wells. Data analyzed with unpaired T -test comparing avelumab vs isotype control; ** p

Techniques Used: ADCC Assay, Expressing, Selection, Purification, In Vitro

Baseline (pre-treatment) expression of PD-L1 as a percentage of parental classic subset. a Representative flow cytometry plots of PD-L1 expression in CD4 + T cells, B cells, cDC, and MDSC. b In 28 patients prior to avelumab therapy, expression of PD-L1 was measured by flow cytometry for nine classic subsets as a percentage of total PBMC, with graphs displaying median and interquartile range
Figure Legend Snippet: Baseline (pre-treatment) expression of PD-L1 as a percentage of parental classic subset. a Representative flow cytometry plots of PD-L1 expression in CD4 + T cells, B cells, cDC, and MDSC. b In 28 patients prior to avelumab therapy, expression of PD-L1 was measured by flow cytometry for nine classic subsets as a percentage of total PBMC, with graphs displaying median and interquartile range

Techniques Used: Expressing, Flow Cytometry, Cytometry

Baseline (pre-treatment) expression of PD-L1 as a percentage of total PBMC. In 28 patients prior to avelumab therapy, expression of PD-L1 was measured by flow cytometry for nine classic subsets as a percentage of total PBMC, with graphs displaying median and interquartile range
Figure Legend Snippet: Baseline (pre-treatment) expression of PD-L1 as a percentage of total PBMC. In 28 patients prior to avelumab therapy, expression of PD-L1 was measured by flow cytometry for nine classic subsets as a percentage of total PBMC, with graphs displaying median and interquartile range

Techniques Used: Expressing, Flow Cytometry, Cytometry

37) Product Images from "Tumor invasion in draining lymph nodes is associated with Treg accumulation in breast cancer patients"

Article Title: Tumor invasion in draining lymph nodes is associated with Treg accumulation in breast cancer patients

Journal: Nature Communications

doi: 10.1038/s41467-020-17046-2

Clinical outcome of differentially expressed genes normalized to FOXP3 expression. a – h OS (upper panels) and DFS (lower panels) of patients with breast cancer stratified by “high” or “low” median intensity of the expression level of CD79, TNFRSF13B, “CD80 , CCR8, and HAVCR2 signature” and CD80 normalized to FOXP3 expression, in TCGA breast cancer data set. i Representative flow cytometric analysis of CD80 expression on Tconv and Treg cells from HD PMBCs (left panels) or tumor (right panels) and representative histogram of CD80 MFI levels in the different subpopulations. j Frequency of CD80+ CD4+ Tconvs or Tregs in HD PBMCs ( N = 2) or tumors CD45+ cells ( N = 8). PBMC-Treg vs tumor-Treg p = 0.044. Mann–Whitney test. Tumor-Tconv vs tumor-Treg p = 0.0078. Wilcoxon matched-pairs signed rank test, * p
Figure Legend Snippet: Clinical outcome of differentially expressed genes normalized to FOXP3 expression. a – h OS (upper panels) and DFS (lower panels) of patients with breast cancer stratified by “high” or “low” median intensity of the expression level of CD79, TNFRSF13B, “CD80 , CCR8, and HAVCR2 signature” and CD80 normalized to FOXP3 expression, in TCGA breast cancer data set. i Representative flow cytometric analysis of CD80 expression on Tconv and Treg cells from HD PMBCs (left panels) or tumor (right panels) and representative histogram of CD80 MFI levels in the different subpopulations. j Frequency of CD80+ CD4+ Tconvs or Tregs in HD PBMCs ( N = 2) or tumors CD45+ cells ( N = 8). PBMC-Treg vs tumor-Treg p = 0.044. Mann–Whitney test. Tumor-Tconv vs tumor-Treg p = 0.0078. Wilcoxon matched-pairs signed rank test, * p

Techniques Used: Expressing, MANN-WHITNEY

38) Product Images from "Regulatory CD4 T cells inhibit HIV-1 expression of other CD4 T cell subsets via interactions with cell surface regulatory proteins"

Article Title: Regulatory CD4 T cells inhibit HIV-1 expression of other CD4 T cell subsets via interactions with cell surface regulatory proteins

Journal: Virology

doi: 10.1016/j.virol.2017.12.036

CD4 Tregs from HIV-1/AIDS patients express lower levels of HIV-1 DNA and viral mRNA than Tconvs and Tmems ex vivo . (Left panels) CD4 T cell populations were sorted from PBMC of newly infected (patient A) and chronically HIV-1 infected (patient B) individuals. Bar graphs depict HIV-1 total DNA (top), gag mRNA (middle), and host FoxP3 mRNA (bottom) for sorted Tregs (Tr), Tconvs (Tc), Tmems (Tm), and Tnaives (Tn) populations. Levels were measured in triplicate and the mean values are shown.
Figure Legend Snippet: CD4 Tregs from HIV-1/AIDS patients express lower levels of HIV-1 DNA and viral mRNA than Tconvs and Tmems ex vivo . (Left panels) CD4 T cell populations were sorted from PBMC of newly infected (patient A) and chronically HIV-1 infected (patient B) individuals. Bar graphs depict HIV-1 total DNA (top), gag mRNA (middle), and host FoxP3 mRNA (bottom) for sorted Tregs (Tr), Tconvs (Tc), Tmems (Tm), and Tnaives (Tn) populations. Levels were measured in triplicate and the mean values are shown.

Techniques Used: Ex Vivo, Infection

39) Product Images from "The Acute Environment, Rather than T Cell Subset Pre-Commitment, Regulates Expression of the Human T Cell Cytokine Amphiregulin"

Article Title: The Acute Environment, Rather than T Cell Subset Pre-Commitment, Regulates Expression of the Human T Cell Cytokine Amphiregulin

Journal: PLoS ONE

doi: 10.1371/journal.pone.0039072

Several human CD4 T cell subsets can produce AR. (A) Allogeneic Th1 and Th2 cell lines from three subjects were stimulated with PMA + ionomycin for 6 hours. The percentage of cells expressing IFNγ, IL-4, and AR was analyzed by ICS. (B) The expression of AR and other cytokines was measured in SEB-stimulated PBMC from four subjects by ICS, calculating the frequencies of single cytokine producers, and all possible combinations of double-producers, among the CD154+ CD4+ T cells. The figure shows the ratio between the observed frequencies of double-producing T cells for each cytokine pair, and the expected frequencies (calculated as the product of the individual frequencies for each cytokine). Values represent the ratios for the double-producer combination defined by the row and column labels. Ratios above or below 1 are indicated by solid or open symbols, respectively. (C) IL-4, IFNγ and IL-2 mRNA levels were measured by RT-PCR in the sorted populations described in Figure 4C . (D) PBMC were treated with influenza H1N1 peptides or tetanus (five subjects each), or the allergens Fel d1 (solid symbols) or Der p1 (open symbols)(three subjects each). The numbers of memory CD4 T cells expressing AR and other cytokines were measured by ICS. The backgrounds (no antigen) have been subtracted. Each symbol represents one individual and the filled bar is the mean of all tested subjects. (E) CD69+ CD4+ T cells (Control_CD69+) were sorted from PBMC incubated in medium alone. CD69+IFNγ+ and CD69+IFNγ- CD4 T cells were sorted from influenza peptide-treated PBMC using the cytokine secretion assay. The mRNA levels of IFNγ and AR were measured by RT-PCR. Results in (A-C) are representative of at least three experiments, (D) represents two experiments using a total of 5 independent subjects, and (E) represents two experiments.
Figure Legend Snippet: Several human CD4 T cell subsets can produce AR. (A) Allogeneic Th1 and Th2 cell lines from three subjects were stimulated with PMA + ionomycin for 6 hours. The percentage of cells expressing IFNγ, IL-4, and AR was analyzed by ICS. (B) The expression of AR and other cytokines was measured in SEB-stimulated PBMC from four subjects by ICS, calculating the frequencies of single cytokine producers, and all possible combinations of double-producers, among the CD154+ CD4+ T cells. The figure shows the ratio between the observed frequencies of double-producing T cells for each cytokine pair, and the expected frequencies (calculated as the product of the individual frequencies for each cytokine). Values represent the ratios for the double-producer combination defined by the row and column labels. Ratios above or below 1 are indicated by solid or open symbols, respectively. (C) IL-4, IFNγ and IL-2 mRNA levels were measured by RT-PCR in the sorted populations described in Figure 4C . (D) PBMC were treated with influenza H1N1 peptides or tetanus (five subjects each), or the allergens Fel d1 (solid symbols) or Der p1 (open symbols)(three subjects each). The numbers of memory CD4 T cells expressing AR and other cytokines were measured by ICS. The backgrounds (no antigen) have been subtracted. Each symbol represents one individual and the filled bar is the mean of all tested subjects. (E) CD69+ CD4+ T cells (Control_CD69+) were sorted from PBMC incubated in medium alone. CD69+IFNγ+ and CD69+IFNγ- CD4 T cells were sorted from influenza peptide-treated PBMC using the cytokine secretion assay. The mRNA levels of IFNγ and AR were measured by RT-PCR. Results in (A-C) are representative of at least three experiments, (D) represents two experiments using a total of 5 independent subjects, and (E) represents two experiments.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation

Both naïve and memory human CD4 T cells expressed AR during TCR activation. (A) PBMC were treated with medium alone or allogeneic EBV-transformed B cells for 10 hours and analyzed by ICS. The gating strategy to identify activated CD4+ and CD8+ T cells is shown. (B) AR, IL-2, IFNγ or IL-4 expression was measured in four subjects in CD45RA+ (open) and CD45RA- (solid) CD4+ and CD8+ T cells after allogeneic EBV-transformed B cell stimulation. Background values have been subtracted. (C) PBMC were treated with medium alone or SEB in the presence of TAPI-1 for 8 hours. Then six populations were sorted based on surface AR, CD69 and CD45RA expression (left). AR mRNA in each population was measured by RT-PCR (right). Results in (A) and (B) represent at least three experiments, (C) represents two experiments.
Figure Legend Snippet: Both naïve and memory human CD4 T cells expressed AR during TCR activation. (A) PBMC were treated with medium alone or allogeneic EBV-transformed B cells for 10 hours and analyzed by ICS. The gating strategy to identify activated CD4+ and CD8+ T cells is shown. (B) AR, IL-2, IFNγ or IL-4 expression was measured in four subjects in CD45RA+ (open) and CD45RA- (solid) CD4+ and CD8+ T cells after allogeneic EBV-transformed B cell stimulation. Background values have been subtracted. (C) PBMC were treated with medium alone or SEB in the presence of TAPI-1 for 8 hours. Then six populations were sorted based on surface AR, CD69 and CD45RA expression (left). AR mRNA in each population was measured by RT-PCR (right). Results in (A) and (B) represent at least three experiments, (C) represents two experiments.

Techniques Used: Activation Assay, Transformation Assay, Expressing, Cell Stimulation, Reverse Transcription Polymerase Chain Reaction

Release of AR from the T cell surface was blocked by the ADAM17 inhibitor TAPI-1. (A) PBMC were stimulated with SEB in the presence or absence of TAPI-1 for variable times. After cell surface staining of AR, the percentage of CD69+AR+ cells within CD4 and CD8 T cells was analyzed. (B) Purified CD4 T cells were treated with medium alone or anti-CD3/CD28 beads with or without TAPI-1 for 24 hours. The concentration of AR in the supernatant was measured by ELISA. All results are representative of at least three experiments.
Figure Legend Snippet: Release of AR from the T cell surface was blocked by the ADAM17 inhibitor TAPI-1. (A) PBMC were stimulated with SEB in the presence or absence of TAPI-1 for variable times. After cell surface staining of AR, the percentage of CD69+AR+ cells within CD4 and CD8 T cells was analyzed. (B) Purified CD4 T cells were treated with medium alone or anti-CD3/CD28 beads with or without TAPI-1 for 24 hours. The concentration of AR in the supernatant was measured by ELISA. All results are representative of at least three experiments.

Techniques Used: Staining, Purification, Concentration Assay, Enzyme-linked Immunosorbent Assay

TCR activation induced AR expression in human PBMC T cells. (A) PBMC were treated as indicated and analyzed by ICS. The upper panels show the gating strategy to identify activated (CD69+) CD4 or CD8 T cells expressing AR. The lower panels show the induction of AR by different stimuli in CD4 or CD8 T cells. (B) AR and IL-2 mRNA were measured by RT-PCR in purified CD4 and CD8 T cells after activation by anti-CD3+anti-CD28 beads. Results in (A) and (B) are representative of at least three experiments.
Figure Legend Snippet: TCR activation induced AR expression in human PBMC T cells. (A) PBMC were treated as indicated and analyzed by ICS. The upper panels show the gating strategy to identify activated (CD69+) CD4 or CD8 T cells expressing AR. The lower panels show the induction of AR by different stimuli in CD4 or CD8 T cells. (B) AR and IL-2 mRNA were measured by RT-PCR in purified CD4 and CD8 T cells after activation by anti-CD3+anti-CD28 beads. Results in (A) and (B) are representative of at least three experiments.

Techniques Used: Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Purification

TCR and cAMP synergize to induce AR production in human CD4 T cells. Purified CD4 T cells were incubated with or without TCR stimulation (anti-CD3/CD28 beads) and the cAMP agonist. (A) AR and HB-EGF mRNA expression was measured by RT-PCR. (B) The concentrations of AR in the supernatant and cell lysates were measured by ELISA. (C) Enriched CD45RA+CD45RO- (naïve) and CD45RA-CD45RO+ (memory) CD4 T cells were treated with medium alone, or anti-CD3/CD28 beads in the presence or absence of cAMP agonist (1 ∼ 1000 µM). The concentration of AR in the supernatant at 24 hours was measured by ELISA. (D) Purified CD4 T cells were treated with medium alone, or anti-CD3/CD28 beads in the presence or absence of the cAMP-modifying agents shown. RNA was extracted at 4 hours, and AR mRNA was measured by RT-PCR. The concentration of AR in the 24-hour supernatant was measured by ELISA. (E) PBMC were treated with anti-CD3+ anti-CD28 antibodies in the presence or absence of cAMP agonist or antagonist for 8 hours. CD4 T cells were purified by cell sorting and RNA was extracted. The mRNA levels of AR and other cytokines were measured by RT-PCR. All results are representative of at least three experiments.
Figure Legend Snippet: TCR and cAMP synergize to induce AR production in human CD4 T cells. Purified CD4 T cells were incubated with or without TCR stimulation (anti-CD3/CD28 beads) and the cAMP agonist. (A) AR and HB-EGF mRNA expression was measured by RT-PCR. (B) The concentrations of AR in the supernatant and cell lysates were measured by ELISA. (C) Enriched CD45RA+CD45RO- (naïve) and CD45RA-CD45RO+ (memory) CD4 T cells were treated with medium alone, or anti-CD3/CD28 beads in the presence or absence of cAMP agonist (1 ∼ 1000 µM). The concentration of AR in the supernatant at 24 hours was measured by ELISA. (D) Purified CD4 T cells were treated with medium alone, or anti-CD3/CD28 beads in the presence or absence of the cAMP-modifying agents shown. RNA was extracted at 4 hours, and AR mRNA was measured by RT-PCR. The concentration of AR in the 24-hour supernatant was measured by ELISA. (E) PBMC were treated with anti-CD3+ anti-CD28 antibodies in the presence or absence of cAMP agonist or antagonist for 8 hours. CD4 T cells were purified by cell sorting and RNA was extracted. The mRNA levels of AR and other cytokines were measured by RT-PCR. All results are representative of at least three experiments.

Techniques Used: Purification, Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Concentration Assay, FACS

40) Product Images from "Recombinant ESAT-6-CFP10 Fusion Protein Induction of Th1/Th2 Cytokines and FoxP3 Expressing Treg Cells in Pulmonary TB"

Article Title: Recombinant ESAT-6-CFP10 Fusion Protein Induction of Th1/Th2 Cytokines and FoxP3 Expressing Treg Cells in Pulmonary TB

Journal: PLoS ONE

doi: 10.1371/journal.pone.0068121

ESAT-6/CFP-10 induced IFN-γ responses in tuberculosis patients and healthy M.bovis BCG vaccinated donors. PBMC obtained from 10 active TB patients and 17 healthy donors were cultured in the presence of ESAT-6/CFP-10 and PHA for 6 days. Cells were then stained to detect the presence of intracellular IFN-γ. The figure represents the overall summary of ESAT-6/CFP-10 (A B) and PHA responses (C D) in CD4 + (A C) and CD8 + (B D) T cells. The bars represent the means and the error bars are 95% CI. P-values were obtained from the unpaired t-test for comparison of healthy donors with untreated TB patients and the repeated measures one-way ANOVA with Sidak’s multiple comparison test was used for comparison of longitudinal data with the baseline results in patients (**P
Figure Legend Snippet: ESAT-6/CFP-10 induced IFN-γ responses in tuberculosis patients and healthy M.bovis BCG vaccinated donors. PBMC obtained from 10 active TB patients and 17 healthy donors were cultured in the presence of ESAT-6/CFP-10 and PHA for 6 days. Cells were then stained to detect the presence of intracellular IFN-γ. The figure represents the overall summary of ESAT-6/CFP-10 (A B) and PHA responses (C D) in CD4 + (A C) and CD8 + (B D) T cells. The bars represent the means and the error bars are 95% CI. P-values were obtained from the unpaired t-test for comparison of healthy donors with untreated TB patients and the repeated measures one-way ANOVA with Sidak’s multiple comparison test was used for comparison of longitudinal data with the baseline results in patients (**P

Techniques Used: Cell Culture, Staining

Gating strategy and results of T cell proliferation assay. Freshly isolated PBMC from 10 TB patients and 10 healthy donors were stained with 5 µM of CFDA-SE and then cultured in the presence of rESAT-6-CFP10 fusion protein (5 µg/ml), PHA (5 µg/ml) or no antigen for 6 days. Harvested cells were stained with anti-CD8-PECy7. Live lymphocytes were gated using the forward and side scatter characteristics to include both resting and dividing cells (R1). CD8+ and CD8- Cells (R3 and R2) were then obtained from the lymphocyte gate. CFSE staining in each cell population was obtained and the different generation of cells were gated R4 to R10. The Figure 5A is representative sample from a control subject. With reference to Figure 5A above, T cell proliferation for 10 TB patients is shown ( Figure 5B ). The bars represent the mean PI (± SE) for CD8 + and CD8 − T cells after subtracting the baseline proliferation. Abbreviations are described in Figure 1 .
Figure Legend Snippet: Gating strategy and results of T cell proliferation assay. Freshly isolated PBMC from 10 TB patients and 10 healthy donors were stained with 5 µM of CFDA-SE and then cultured in the presence of rESAT-6-CFP10 fusion protein (5 µg/ml), PHA (5 µg/ml) or no antigen for 6 days. Harvested cells were stained with anti-CD8-PECy7. Live lymphocytes were gated using the forward and side scatter characteristics to include both resting and dividing cells (R1). CD8+ and CD8- Cells (R3 and R2) were then obtained from the lymphocyte gate. CFSE staining in each cell population was obtained and the different generation of cells were gated R4 to R10. The Figure 5A is representative sample from a control subject. With reference to Figure 5A above, T cell proliferation for 10 TB patients is shown ( Figure 5B ). The bars represent the mean PI (± SE) for CD8 + and CD8 − T cells after subtracting the baseline proliferation. Abbreviations are described in Figure 1 .

Techniques Used: Proliferation Assay, Isolation, Staining, Cell Culture

rESAT-6-CFP10-induced changes in the ratio of the proportion of T cells expressing IFN-γ/IL-4 in tuberculosis patients. PBMC obtained from 10 active TB patients and were cultured in the presence of rESAT-6-CFP10. Cells were then stained to detect the presence of intracellular IFN-γ and IL-4. The ratio of the proportion of CD4 + (A) and CD8 + (B) T cells expressing IFN-γ/IL-4 for individual patients at diagnosis and during follow up are represented. The significant p-values obtained from the Friedman’s test with Dunn’s post-test correction for the mean ratios were 0.0017 at T2 v T0 and 0.001 at T3 v T0 in CD4 + T cells and 0.0022 at T3 v T0 in CD8 + T cells. Abbreviations are described in Figure 1 .
Figure Legend Snippet: rESAT-6-CFP10-induced changes in the ratio of the proportion of T cells expressing IFN-γ/IL-4 in tuberculosis patients. PBMC obtained from 10 active TB patients and were cultured in the presence of rESAT-6-CFP10. Cells were then stained to detect the presence of intracellular IFN-γ and IL-4. The ratio of the proportion of CD4 + (A) and CD8 + (B) T cells expressing IFN-γ/IL-4 for individual patients at diagnosis and during follow up are represented. The significant p-values obtained from the Friedman’s test with Dunn’s post-test correction for the mean ratios were 0.0017 at T2 v T0 and 0.001 at T3 v T0 in CD4 + T cells and 0.0022 at T3 v T0 in CD8 + T cells. Abbreviations are described in Figure 1 .

Techniques Used: Expressing, Cell Culture, Staining

rESAT-6-CFP10 induced IL-4 responses in tuberculosis patients and healthy M.bovis BCG vaccinated healthy donors. PBMC obtained from 10 active TB patients and 17 healthy donors were cultured in the presence of rESAT-6-CFP10 or PHA for 6 days. Cells were then stained to detect the presence of intracellular IL-4. The figure represents the overall summary for rESAT-6-CFP10 (A B) and PHA responses (C D) in CD4 + (A C) and CD8 + (B D) T cells. The bars represent the means and the error bars are 95% CI. P-values were obtained from the Mann-Whitney test for comparison of healthy donors with T0 and comparisons were made between T0 and other time points for TB patients using the non-parametric repeated measures one-way ANOVA (Friedman’s test) with Dunn’s multiple comparison (**P
Figure Legend Snippet: rESAT-6-CFP10 induced IL-4 responses in tuberculosis patients and healthy M.bovis BCG vaccinated healthy donors. PBMC obtained from 10 active TB patients and 17 healthy donors were cultured in the presence of rESAT-6-CFP10 or PHA for 6 days. Cells were then stained to detect the presence of intracellular IL-4. The figure represents the overall summary for rESAT-6-CFP10 (A B) and PHA responses (C D) in CD4 + (A C) and CD8 + (B D) T cells. The bars represent the means and the error bars are 95% CI. P-values were obtained from the Mann-Whitney test for comparison of healthy donors with T0 and comparisons were made between T0 and other time points for TB patients using the non-parametric repeated measures one-way ANOVA (Friedman’s test) with Dunn’s multiple comparison (**P

Techniques Used: Cell Culture, Staining, MANN-WHITNEY

rESAT-6-CFP10-induced regulatory T cells in TB patients and healthy donors. Freshly isolated PBMC from 10 TB patients were cultured in the presence of ESAT-6-CFP10 or without antigen for 6 days. Harvested cells were then stained for the expression of CD25 and Foxp3 within the CD4 − (B D) and CD4 + (A C) T cell populations. The figure illustrates the mean proportions (horizontal lines) of CD4 + and CD4 − T cells expressing both CD25 and FoxP3 (A B) or CD25 with high fluorescent intensity (CD25 hi ) and Foxp3 (C D) after subtracting the responses obtained in unstimulated cultures. The dots are the individual responses and the error bars represent the standard errors of the mean. The repeated measures one-way ANOVA with Sidak’s multiple comparison tests was used for comparison of longitudinal data with the baseline results in patients. The Mann Whitney p-value was used in the comparison of baseline results from TB patients with healthy donors (**P
Figure Legend Snippet: rESAT-6-CFP10-induced regulatory T cells in TB patients and healthy donors. Freshly isolated PBMC from 10 TB patients were cultured in the presence of ESAT-6-CFP10 or without antigen for 6 days. Harvested cells were then stained for the expression of CD25 and Foxp3 within the CD4 − (B D) and CD4 + (A C) T cell populations. The figure illustrates the mean proportions (horizontal lines) of CD4 + and CD4 − T cells expressing both CD25 and FoxP3 (A B) or CD25 with high fluorescent intensity (CD25 hi ) and Foxp3 (C D) after subtracting the responses obtained in unstimulated cultures. The dots are the individual responses and the error bars represent the standard errors of the mean. The repeated measures one-way ANOVA with Sidak’s multiple comparison tests was used for comparison of longitudinal data with the baseline results in patients. The Mann Whitney p-value was used in the comparison of baseline results from TB patients with healthy donors (**P

Techniques Used: Isolation, Cell Culture, Staining, Expressing, MANN-WHITNEY

An illustration of the trend in rESAT-6-CFP10-induced regulatory T cell expression and IFN-γ/IL-4 ratios in the intensive phase of TB treatment. Freshly isolated PBMC from 10 TB patients were cultured in the presence of ESAT-6-CFP10 or without antigen for 6 days. Harvested cells were then stained for the expression of CD25, Foxp3, IFN-γ and IL-4. The mean regulatory T cell expression is plotted against the mean of the ratio of IFN-γ/IL-4 expressing T cells at diagnosis and during follow up of TB patients. Abbreviations are explained in Figure 1 .
Figure Legend Snippet: An illustration of the trend in rESAT-6-CFP10-induced regulatory T cell expression and IFN-γ/IL-4 ratios in the intensive phase of TB treatment. Freshly isolated PBMC from 10 TB patients were cultured in the presence of ESAT-6-CFP10 or without antigen for 6 days. Harvested cells were then stained for the expression of CD25, Foxp3, IFN-γ and IL-4. The mean regulatory T cell expression is plotted against the mean of the ratio of IFN-γ/IL-4 expressing T cells at diagnosis and during follow up of TB patients. Abbreviations are explained in Figure 1 .

Techniques Used: Expressing, Isolation, Cell Culture, Staining

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Flow Cytometry:

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Cytometry:

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Infection:

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Cell Differentiation:

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Incubation:

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Expressing:

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Staining:

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Article Snippet: .. Flow cytometry For detection of Treg cells, PBMCs were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD4, phycoerythrin (PE)-Cy5-conjugated anti-CD25, and PE-conjugated anti-CD127 (BD Pharmingen, San Jose, CA, USA) according to the manufacturer’s protocol. ..

Article Title: CD8+ T cells from HLA-B*57 elite suppressors effectively suppress replication of HIV-1 escape mutants
Article Snippet: .. Intracellular cytokine analysis 0.5 × 106 unstimulated or stimulated PBMCs (from above) were restimulated with peptide (2 ug/mL), anti-CD28, and anti-CD49d in the presence of GolgiStop and GolgiPlug (BD) for 12 h. After staining with CD3 PE and CD8 APC-H7 (BD), cells were fixed and permeablized with Cytoperm/Cytofix Kit (BD). .. Cytokines were stained using IFN-γ PerCP-Cy5.5 (BD) and Perforin FITC (Cell Sciences).

Article Title: A Single HIV-1 Cluster and a Skewed Immune Homeostasis Drive the Early Spread of HIV among Resting CD4+ Cell Subsets within One Month Post-Infection
Article Snippet: .. CD4 T Cell Differentiation Analysis and Sorting PBMC cryopreserved and stored in liquid nitrogen, with more than 80% viability after thawing, were sorted as live monocytes (CD3−CD4+) or activated and resting CD3+CD4+ T cells on a 5-laser FACS ARIA (Becton Dickinson) on the CyPS platform (UPMC) after staining with the following combination: Live-Dead Fixable Aqua (Life Technologies), CD3-Pacific Blue (UCHT1), CD4-AlexaFluor700 (RPA-T4), CCR7-PE Cyanine7 (3D12), CD27-APC (L128), CD69-FITC (L78), HLA-DR-FITC (L243) and CD127-PE (IL7R-M21) from BD Pharmingen, CD45RA-ECD (2H4) and CD25-FITC (B1.49.9) from Beckman Coulter. .. Resting CD4 T cells (CD25−CD69−HLADR−) were further sorted as: naive (TN, CD45RA+CCR7+CD27+), central-memory (TCM, CD45RA−CCR7+CD27+), transitional-memory (TTM, CD45RA−CCR7−CD27+), and effector-memory cells (TEM, CD45RA−CCR7−CD27−) ( ).

FACS:

Article Title: Preexisting Virus-Specific T Lymphocytes-Mediated Enhancement of Adenovirus Infections to Human Blood CD14+ Cells
Article Snippet: .. For detecting the expression of EGFP reporter gene in different cell population, the infected PBMCs were incubated with corresponding fluorescent-labeled monoclonal antibodies (CD3-APC, CD3-PE, CD3-PerCP, CD14-APC, CD14-PE, CD19-PE-cy5, CD56-PE, CD27-APC, CD95-PE, HLADR-APC, Ki67-PE, 7-AAD, BD Pharmingen, San Diego, CA, USA) and CD38-FITC (STEMCELL Technologies, Vancouver, Canada), Integrinβ5-PE (eBioscience, San Diego, CA, USA), and then detected with a BD FACS LSR Fortessa flow cytometer (BD Biosciences, San Diego, CA, USA). .. For detecting the expression of SEAP reporter gene, PBMCs were seeded at 5 × 105 cells per well in 96-well plates, and then incubated with the indicated dosage of Ad-SEAP for 24–48 h at 37 °C in 5% CO2 incubator.

Article Title: A Single HIV-1 Cluster and a Skewed Immune Homeostasis Drive the Early Spread of HIV among Resting CD4+ Cell Subsets within One Month Post-Infection
Article Snippet: .. CD4 T Cell Differentiation Analysis and Sorting PBMC cryopreserved and stored in liquid nitrogen, with more than 80% viability after thawing, were sorted as live monocytes (CD3−CD4+) or activated and resting CD3+CD4+ T cells on a 5-laser FACS ARIA (Becton Dickinson) on the CyPS platform (UPMC) after staining with the following combination: Live-Dead Fixable Aqua (Life Technologies), CD3-Pacific Blue (UCHT1), CD4-AlexaFluor700 (RPA-T4), CCR7-PE Cyanine7 (3D12), CD27-APC (L128), CD69-FITC (L78), HLA-DR-FITC (L243) and CD127-PE (IL7R-M21) from BD Pharmingen, CD45RA-ECD (2H4) and CD25-FITC (B1.49.9) from Beckman Coulter. .. Resting CD4 T cells (CD25−CD69−HLADR−) were further sorted as: naive (TN, CD45RA+CCR7+CD27+), central-memory (TCM, CD45RA−CCR7+CD27+), transitional-memory (TTM, CD45RA−CCR7−CD27+), and effector-memory cells (TEM, CD45RA−CCR7−CD27−) ( ).

Recombinase Polymerase Amplification:

Article Title: Blocking TLR7- and TLR9-mediated IFN-? Production by Plasmacytoid Dendritic Cells Does Not Diminish Immune Activation in Early SIV Infection
Article Snippet: .. Flow cytometry The following monoclonal antibodies were used to label PBMC and lymph node cell suspensions and were purchased from BD Biosciences unless otherwise noted: CD3 (clone SP34-2), CD20 (eBiosciences, 2H7), CD14 (MøP9), CD123 (7G3), CD11c (S-HCL-3), HLA-DR (L243 or G46-6), CD163 (GHI/61), CCR7 (150503, R & D Systems), CD4 (L200), CD8 (RPA-T8), CD28 (CD28.2), CD38 (AT-1, Stem cell Technologies), CD95 (DX2), IRF-7 (H-246, Santa Cruz Biotechnology) and Ki67 (B56). .. Flow cytometric analysis and determination of blood pDC and CD4+ T cell counts were done as described , .

Article Title: A Single HIV-1 Cluster and a Skewed Immune Homeostasis Drive the Early Spread of HIV among Resting CD4+ Cell Subsets within One Month Post-Infection
Article Snippet: .. CD4 T Cell Differentiation Analysis and Sorting PBMC cryopreserved and stored in liquid nitrogen, with more than 80% viability after thawing, were sorted as live monocytes (CD3−CD4+) or activated and resting CD3+CD4+ T cells on a 5-laser FACS ARIA (Becton Dickinson) on the CyPS platform (UPMC) after staining with the following combination: Live-Dead Fixable Aqua (Life Technologies), CD3-Pacific Blue (UCHT1), CD4-AlexaFluor700 (RPA-T4), CCR7-PE Cyanine7 (3D12), CD27-APC (L128), CD69-FITC (L78), HLA-DR-FITC (L243) and CD127-PE (IL7R-M21) from BD Pharmingen, CD45RA-ECD (2H4) and CD25-FITC (B1.49.9) from Beckman Coulter. .. Resting CD4 T cells (CD25−CD69−HLADR−) were further sorted as: naive (TN, CD45RA+CCR7+CD27+), central-memory (TCM, CD45RA−CCR7+CD27+), transitional-memory (TTM, CD45RA−CCR7−CD27+), and effector-memory cells (TEM, CD45RA−CCR7−CD27−) ( ).

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    Becton Dickinson pbmcs
    Intracellular <t>cytokine</t> staining of CP CD8 + T cells. A-D: CD8 + T cells of HLA-B*57 positive CP were either freshly isolated (left) or stimulated with either wild type (center) or escape variant (right) HLA-B*57-restricted Gag peptides for 7 days. CPs were either on suppressive HAART regiments with undetectable viral loads (circles), were on HAART regimens but recently had detectable levels of viremia (triangles), or were not on HAART and had high levels of viremia (diamond). Cells from each group underwent an overnight stimulation with individual peptides. Percentage of CD8 + T cells expressing IFN-γ when restimulated with TW10 (A) , KF11 (B) , and IW9 (C) in blue, or the escape mutant variant peptide containing T242N/G248A (A) , A163S (B) , and I147L (C) mutations in red is shown. E-H : Percentage of CD8 + T cells expressing both IFN-γ and perforin after overnight stimulation with TW10 (E) , KF11 (F) , and IW9 (G) in blue, or the escape mutant variant peptide containing T242N/G248A (E) , A163S (F) , and I147L (G) mutations in red is shown. D and H show CD8 + T cells that express IFN-γ or co-express IFN-γ and perforin when <t>PBMCs</t> were stimulated overnight with Gag 263-272 (KK10, HLA-B*27 + peptide). Black asterisks indicate statistically significant difference (P
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    Intracellular cytokine staining of CP CD8 + T cells. A-D: CD8 + T cells of HLA-B*57 positive CP were either freshly isolated (left) or stimulated with either wild type (center) or escape variant (right) HLA-B*57-restricted Gag peptides for 7 days. CPs were either on suppressive HAART regiments with undetectable viral loads (circles), were on HAART regimens but recently had detectable levels of viremia (triangles), or were not on HAART and had high levels of viremia (diamond). Cells from each group underwent an overnight stimulation with individual peptides. Percentage of CD8 + T cells expressing IFN-γ when restimulated with TW10 (A) , KF11 (B) , and IW9 (C) in blue, or the escape mutant variant peptide containing T242N/G248A (A) , A163S (B) , and I147L (C) mutations in red is shown. E-H : Percentage of CD8 + T cells expressing both IFN-γ and perforin after overnight stimulation with TW10 (E) , KF11 (F) , and IW9 (G) in blue, or the escape mutant variant peptide containing T242N/G248A (E) , A163S (F) , and I147L (G) mutations in red is shown. D and H show CD8 + T cells that express IFN-γ or co-express IFN-γ and perforin when PBMCs were stimulated overnight with Gag 263-272 (KK10, HLA-B*27 + peptide). Black asterisks indicate statistically significant difference (P

    Journal: Retrovirology

    Article Title: CD8+ T cells from HLA-B*57 elite suppressors effectively suppress replication of HIV-1 escape mutants

    doi: 10.1186/1742-4690-10-152

    Figure Lengend Snippet: Intracellular cytokine staining of CP CD8 + T cells. A-D: CD8 + T cells of HLA-B*57 positive CP were either freshly isolated (left) or stimulated with either wild type (center) or escape variant (right) HLA-B*57-restricted Gag peptides for 7 days. CPs were either on suppressive HAART regiments with undetectable viral loads (circles), were on HAART regimens but recently had detectable levels of viremia (triangles), or were not on HAART and had high levels of viremia (diamond). Cells from each group underwent an overnight stimulation with individual peptides. Percentage of CD8 + T cells expressing IFN-γ when restimulated with TW10 (A) , KF11 (B) , and IW9 (C) in blue, or the escape mutant variant peptide containing T242N/G248A (A) , A163S (B) , and I147L (C) mutations in red is shown. E-H : Percentage of CD8 + T cells expressing both IFN-γ and perforin after overnight stimulation with TW10 (E) , KF11 (F) , and IW9 (G) in blue, or the escape mutant variant peptide containing T242N/G248A (E) , A163S (F) , and I147L (G) mutations in red is shown. D and H show CD8 + T cells that express IFN-γ or co-express IFN-γ and perforin when PBMCs were stimulated overnight with Gag 263-272 (KK10, HLA-B*27 + peptide). Black asterisks indicate statistically significant difference (P

    Article Snippet: Intracellular cytokine analysis 0.5 × 106 unstimulated or stimulated PBMCs (from above) were restimulated with peptide (2 ug/mL), anti-CD28, and anti-CD49d in the presence of GolgiStop and GolgiPlug (BD) for 12 h. After staining with CD3 PE and CD8 APC-H7 (BD), cells were fixed and permeablized with Cytoperm/Cytofix Kit (BD).

    Techniques: Staining, Isolation, Variant Assay, Expressing, Mutagenesis

    Intracellular cytokine staining of ES CD8 + T cells. A-D: CD8 + T cells of HLA-B*57 positive ES were either freshly isolated (left) or stimulated with either wild type (center) or escape variant (right) HLA-B*57-restricted Gag peptides for 7 days. Cells from each group underwent an overnight stimulation with individual peptides. Percentage of CD8 + T cells expressing IFN-γ when stimulated overnight with TW10 (A) , KF11 (B) , and IW9 (C) in blue, or the escape mutant variant peptide containing T242N/G248A (A) , A163S (B) , and I147L (C) mutations in red is shown. E-H : Percentage of CD8 + T cells expressing both IFN-γ and perforin after restimulation with TW10 (E) , KF11 (F) , and IW9 (B) in blue, or the escape mutant variant peptide containing T242N/G248A (E) , A163S (F) , and I147L (G) in red is shown. D and H show CD8 + T cells that express IFN-γ or co-express IFN-γ and perforin when PBMCs were stimulated overnight with Gag 263-272 (KK10, HLA-B*27 + peptide). Black horizontal bars indicate statistically significant difference (P

    Journal: Retrovirology

    Article Title: CD8+ T cells from HLA-B*57 elite suppressors effectively suppress replication of HIV-1 escape mutants

    doi: 10.1186/1742-4690-10-152

    Figure Lengend Snippet: Intracellular cytokine staining of ES CD8 + T cells. A-D: CD8 + T cells of HLA-B*57 positive ES were either freshly isolated (left) or stimulated with either wild type (center) or escape variant (right) HLA-B*57-restricted Gag peptides for 7 days. Cells from each group underwent an overnight stimulation with individual peptides. Percentage of CD8 + T cells expressing IFN-γ when stimulated overnight with TW10 (A) , KF11 (B) , and IW9 (C) in blue, or the escape mutant variant peptide containing T242N/G248A (A) , A163S (B) , and I147L (C) mutations in red is shown. E-H : Percentage of CD8 + T cells expressing both IFN-γ and perforin after restimulation with TW10 (E) , KF11 (F) , and IW9 (B) in blue, or the escape mutant variant peptide containing T242N/G248A (E) , A163S (F) , and I147L (G) in red is shown. D and H show CD8 + T cells that express IFN-γ or co-express IFN-γ and perforin when PBMCs were stimulated overnight with Gag 263-272 (KK10, HLA-B*27 + peptide). Black horizontal bars indicate statistically significant difference (P

    Article Snippet: Intracellular cytokine analysis 0.5 × 106 unstimulated or stimulated PBMCs (from above) were restimulated with peptide (2 ug/mL), anti-CD28, and anti-CD49d in the presence of GolgiStop and GolgiPlug (BD) for 12 h. After staining with CD3 PE and CD8 APC-H7 (BD), cells were fixed and permeablized with Cytoperm/Cytofix Kit (BD).

    Techniques: Staining, Isolation, Variant Assay, Expressing, Mutagenesis

    Flow cytometry analysis of different subsets of CD4+ T cells. PBMCs were isolated form individual participants and stimulated with, or without, PMA/ionomycin and harvested. The cells were stained with APC-anti-CD4, fixed, and permeabilized, followed by intracellular staining with FITC-anti-IL-17, PE-Cy7-anti-IFNγ, and PE-anti-IL-22 and flow cytometry. Subsequently, the cells were gated first on CD4 + cells for analysis of the frequency of CD4 + IFNγ + and CD4 + IFNγ − cells. The CD4 + IFNγ + cells were further analyzed for CD4 + IFNγ + IL-17 + cells (column I), while the CD4 + IFNγ − cells were further analyzed for CD4 + IFNγ − IL-17 + , CD4 + IFNγ − IL-22 + , and CD4 + IFNγ − IL-17 + IL-22 + cells (column II), followed by quantitative analyses. Data are representative charts or expressed as the mean values of individual participants from sequential experiments. A. Representative charts of flow cytometry analysis; B. Quantitative analysis.

    Journal: PLoS ONE

    Article Title: A High Frequency of Circulating Th22 and Th17 Cells in Patients with New Onset Graves' Disease

    doi: 10.1371/journal.pone.0068446

    Figure Lengend Snippet: Flow cytometry analysis of different subsets of CD4+ T cells. PBMCs were isolated form individual participants and stimulated with, or without, PMA/ionomycin and harvested. The cells were stained with APC-anti-CD4, fixed, and permeabilized, followed by intracellular staining with FITC-anti-IL-17, PE-Cy7-anti-IFNγ, and PE-anti-IL-22 and flow cytometry. Subsequently, the cells were gated first on CD4 + cells for analysis of the frequency of CD4 + IFNγ + and CD4 + IFNγ − cells. The CD4 + IFNγ + cells were further analyzed for CD4 + IFNγ + IL-17 + cells (column I), while the CD4 + IFNγ − cells were further analyzed for CD4 + IFNγ − IL-17 + , CD4 + IFNγ − IL-22 + , and CD4 + IFNγ − IL-17 + IL-22 + cells (column II), followed by quantitative analyses. Data are representative charts or expressed as the mean values of individual participants from sequential experiments. A. Representative charts of flow cytometry analysis; B. Quantitative analysis.

    Article Snippet: Briefly, the stimulated PBMCs were harvested and stained with allophycocyanin (APC)-labeled anti-CD4, fixed with the Perm/Fix solution, and permeabilized, followed by staining with fluorescein isothiocyanate (FITC)-labeled anti–IL-17, PE-Cy7-labeled anti-IFNγ (Becton Dickinson, San Diego, USA), and PE-labeled anti–IL-22 (R & D Systems, Minneapolis, MN, USA).

    Techniques: Flow Cytometry, Cytometry, Isolation, Staining

    CD4 T cell subsets repartition and contribution to the pool of infected cells. A : Monocytes, activated and resting CD4 T-cell (CD4 T Ly) contributions to the pool of infected PBMCs were calculated with the infection level and frequency of each subset. Only significant p values are shown. B : The repartition of resting CD4 T-cell subsets was assessed in twelve acutely HIV-infected individuals (grey) and in ten uninfected individuals (white). The analyzed resting CD4+ subsets are: naive (TN, CD45RA+CCR7+CD27+), central-memory (TCM, CD45RA-CCR7+CD27+), transitional-memory (TTM, CD45RA−CCR7−CD27+) and effector-memory cells (TEM, CD45RA−CCR7−CD27−). Results are expressed as the percentage of resting CD4 T cells. C : Resting CD4 T-cell subset contributions to the pool of infected resting CD4 T cells were calculated with the infection level and frequency of each subset.

    Journal: PLoS ONE

    Article Title: A Single HIV-1 Cluster and a Skewed Immune Homeostasis Drive the Early Spread of HIV among Resting CD4+ Cell Subsets within One Month Post-Infection

    doi: 10.1371/journal.pone.0064219

    Figure Lengend Snippet: CD4 T cell subsets repartition and contribution to the pool of infected cells. A : Monocytes, activated and resting CD4 T-cell (CD4 T Ly) contributions to the pool of infected PBMCs were calculated with the infection level and frequency of each subset. Only significant p values are shown. B : The repartition of resting CD4 T-cell subsets was assessed in twelve acutely HIV-infected individuals (grey) and in ten uninfected individuals (white). The analyzed resting CD4+ subsets are: naive (TN, CD45RA+CCR7+CD27+), central-memory (TCM, CD45RA-CCR7+CD27+), transitional-memory (TTM, CD45RA−CCR7−CD27+) and effector-memory cells (TEM, CD45RA−CCR7−CD27−). Results are expressed as the percentage of resting CD4 T cells. C : Resting CD4 T-cell subset contributions to the pool of infected resting CD4 T cells were calculated with the infection level and frequency of each subset.

    Article Snippet: CD4 T Cell Differentiation Analysis and Sorting PBMC cryopreserved and stored in liquid nitrogen, with more than 80% viability after thawing, were sorted as live monocytes (CD3−CD4+) or activated and resting CD3+CD4+ T cells on a 5-laser FACS ARIA (Becton Dickinson) on the CyPS platform (UPMC) after staining with the following combination: Live-Dead Fixable Aqua (Life Technologies), CD3-Pacific Blue (UCHT1), CD4-AlexaFluor700 (RPA-T4), CCR7-PE Cyanine7 (3D12), CD27-APC (L128), CD69-FITC (L78), HLA-DR-FITC (L243) and CD127-PE (IL7R-M21) from BD Pharmingen, CD45RA-ECD (2H4) and CD25-FITC (B1.49.9) from Beckman Coulter.

    Techniques: Infection, Transmission Electron Microscopy

    TSA, SAHA, and MS-275 decrease TLR9-induced activation of IRF-7 and NF-κB p65 transcription factors. PBMC were incubated with TSA (100 ng/ml) or DMSO (1:1000) for 1 hour, stimulated with HSV-1 (MOI of 1) or IAV (MOI of 2) for 3 hours. Samples were processed using BD Phosflow protocol. PBMC were gated based on SSC vs. FSC-A (Area) and pDC were identified by gating on +HLA-DRhigh/+CD123high events on a HLA-DR APC vs. CD123 PE intensity dot plot, then percent phosphorylated IRF-7+ pDC were gated based on mock sample for HSV-1 or IAV from which percentages were reported. Representative analysis for TSA, SAHA, and MS-275-mediated effect on IAV-mediated IRF-7 and NF-kB p65 phosphorylation in pDC. Red lines represent the position where the gate was placed from which percentages were derived. (A) . Pooled data with HSV-1 stimulation (top panels) and IAV (bottom panels) showing the effect of TSA (100 ng/ml) (B) , MS-275 (5 μM) (C) , and SAHA (500 nM) (D) , or DMSO (1:1000) on the phosphorylation of IRF-7 and NF-kB p65. Data represent the percentage of pIRF-7+ and pNF-κB p65+ pDC with gating based on mock sample. (N = 4-5, 4-5 independent experiments with different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; n.s. = not significant).

    Journal: bioRxiv

    Article Title: HDAC inhibitors decrease TLR7/9-mediated human plasmacytoid dendritic cell activation by interfering with IRF-7 and NF-κB signaling

    doi: 10.1101/2020.05.09.085456

    Figure Lengend Snippet: TSA, SAHA, and MS-275 decrease TLR9-induced activation of IRF-7 and NF-κB p65 transcription factors. PBMC were incubated with TSA (100 ng/ml) or DMSO (1:1000) for 1 hour, stimulated with HSV-1 (MOI of 1) or IAV (MOI of 2) for 3 hours. Samples were processed using BD Phosflow protocol. PBMC were gated based on SSC vs. FSC-A (Area) and pDC were identified by gating on +HLA-DRhigh/+CD123high events on a HLA-DR APC vs. CD123 PE intensity dot plot, then percent phosphorylated IRF-7+ pDC were gated based on mock sample for HSV-1 or IAV from which percentages were reported. Representative analysis for TSA, SAHA, and MS-275-mediated effect on IAV-mediated IRF-7 and NF-kB p65 phosphorylation in pDC. Red lines represent the position where the gate was placed from which percentages were derived. (A) . Pooled data with HSV-1 stimulation (top panels) and IAV (bottom panels) showing the effect of TSA (100 ng/ml) (B) , MS-275 (5 μM) (C) , and SAHA (500 nM) (D) , or DMSO (1:1000) on the phosphorylation of IRF-7 and NF-kB p65. Data represent the percentage of pIRF-7+ and pNF-κB p65+ pDC with gating based on mock sample. (N = 4-5, 4-5 independent experiments with different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; n.s. = not significant).

    Article Snippet: In order to answer this question, PBMC were isolated and pre-treated with TSA or SAHA for 1 hour, then stimulated with HSV-1 for 3 hours, then we utilized BD Phosflow™ assays to assess the phosphorylation status of IRF-7 and NF-κB p65 in the presence of TSA, MS-275, SAHA.

    Techniques: Activation Assay, Incubation, Derivative Assay

    TSA and MS-275 inhibit HSV-1 and IAV-induced total IFN-α production in human pDC. PBMC were isolated and pre-treated with TSA (100 ng/ml) or DMSO vehicle control (1:1000) (A) and MS-275 (5 μM) or DMSO vehicle control (1:530) (B) for 1 hour and then stimulated with HSV-1 (MOI of 1) and IAV (MOI of 2) for 18 hours. Supernatants were collected and tested for IFN-α production by an ELISA assay. (N=4, 4 independent experiments from different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; *p

    Journal: bioRxiv

    Article Title: HDAC inhibitors decrease TLR7/9-mediated human plasmacytoid dendritic cell activation by interfering with IRF-7 and NF-κB signaling

    doi: 10.1101/2020.05.09.085456

    Figure Lengend Snippet: TSA and MS-275 inhibit HSV-1 and IAV-induced total IFN-α production in human pDC. PBMC were isolated and pre-treated with TSA (100 ng/ml) or DMSO vehicle control (1:1000) (A) and MS-275 (5 μM) or DMSO vehicle control (1:530) (B) for 1 hour and then stimulated with HSV-1 (MOI of 1) and IAV (MOI of 2) for 18 hours. Supernatants were collected and tested for IFN-α production by an ELISA assay. (N=4, 4 independent experiments from different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; *p

    Article Snippet: In order to answer this question, PBMC were isolated and pre-treated with TSA or SAHA for 1 hour, then stimulated with HSV-1 for 3 hours, then we utilized BD Phosflow™ assays to assess the phosphorylation status of IRF-7 and NF-κB p65 in the presence of TSA, MS-275, SAHA.

    Techniques: Isolation, Enzyme-linked Immunosorbent Assay

    TSA and MS-275 inhibit the upregulation of pDC maturation markers upon TLR7/9 stimulation, and increase the shedding of the activation marker CD62L. PBMC were incubated with TSA (100 ng/ml), MS-275 (5 μM), or DMSO (1:530) for 1 hour and then stimulated with HSV-1 (MOI of 1), IAV (MOI of 2), or CpG-B (5 μg/ml) for 8 hours. Representative data derived from events first gated on PBMC, then pDC were identified by a BDCA-2 + /CD123 high gate. From this gate, histograms were derived and gates applied based on the unstimulated control in DMSO (Mock) (A) . Surface expression of CD62L (B, top) , CD40 (B, bottom) , CD86 (C, top), CD82 (C, bottom) were measured by flow cytometry and reported as percentage. (N = 4, 4 independent experiments with different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA and Bonferroni’s post test; *p

    Journal: bioRxiv

    Article Title: HDAC inhibitors decrease TLR7/9-mediated human plasmacytoid dendritic cell activation by interfering with IRF-7 and NF-κB signaling

    doi: 10.1101/2020.05.09.085456

    Figure Lengend Snippet: TSA and MS-275 inhibit the upregulation of pDC maturation markers upon TLR7/9 stimulation, and increase the shedding of the activation marker CD62L. PBMC were incubated with TSA (100 ng/ml), MS-275 (5 μM), or DMSO (1:530) for 1 hour and then stimulated with HSV-1 (MOI of 1), IAV (MOI of 2), or CpG-B (5 μg/ml) for 8 hours. Representative data derived from events first gated on PBMC, then pDC were identified by a BDCA-2 + /CD123 high gate. From this gate, histograms were derived and gates applied based on the unstimulated control in DMSO (Mock) (A) . Surface expression of CD62L (B, top) , CD40 (B, bottom) , CD86 (C, top), CD82 (C, bottom) were measured by flow cytometry and reported as percentage. (N = 4, 4 independent experiments with different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA and Bonferroni’s post test; *p

    Article Snippet: In order to answer this question, PBMC were isolated and pre-treated with TSA or SAHA for 1 hour, then stimulated with HSV-1 for 3 hours, then we utilized BD Phosflow™ assays to assess the phosphorylation status of IRF-7 and NF-κB p65 in the presence of TSA, MS-275, SAHA.

    Techniques: Activation Assay, Marker, Incubation, Derivative Assay, Expressing, Flow Cytometry

    TSA and MS-275, but not SAHA, inhibits IAV and HSV-1-mediated IFN-α protein upregulation without decreasing IRF-7 upregulation. PBMC were pre-treated with TSA (100 ng/ml), SAHA (500 nM) and DMSO vehicle control (1:1000) or MS-275 (5 μM) and DMSO (1:530), for 1 hour and then stimulated with HSV-1 (MOI of 1) or IAV (MOI of 2) for 6 hours. IRF-7 (black bars) and intracellular IFN-α (gray bars) protein expression was measured concurrently by intracellular flow cytometry. Representative data showing the effect, on IAV-induced IFN (A, top) and IRF-7 (A, bottom) upregulation in pDC, by TSA, MS-275, and SAHA (A). Cumulative experiments showing the effect on HSV-1 (top) and IAV (bottom)-induced upregulation of IFN-α and IRF-7, by TSA (100 ng/ml) (B) and MS-275 (5 μM) (C) and SAHA (500 nM) (D). (N = 5; 5 independent experiments with different donors for IAV. N = 3; 3 independent experiments with different donors for HSV-1. Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; (* = p

    Journal: bioRxiv

    Article Title: HDAC inhibitors decrease TLR7/9-mediated human plasmacytoid dendritic cell activation by interfering with IRF-7 and NF-κB signaling

    doi: 10.1101/2020.05.09.085456

    Figure Lengend Snippet: TSA and MS-275, but not SAHA, inhibits IAV and HSV-1-mediated IFN-α protein upregulation without decreasing IRF-7 upregulation. PBMC were pre-treated with TSA (100 ng/ml), SAHA (500 nM) and DMSO vehicle control (1:1000) or MS-275 (5 μM) and DMSO (1:530), for 1 hour and then stimulated with HSV-1 (MOI of 1) or IAV (MOI of 2) for 6 hours. IRF-7 (black bars) and intracellular IFN-α (gray bars) protein expression was measured concurrently by intracellular flow cytometry. Representative data showing the effect, on IAV-induced IFN (A, top) and IRF-7 (A, bottom) upregulation in pDC, by TSA, MS-275, and SAHA (A). Cumulative experiments showing the effect on HSV-1 (top) and IAV (bottom)-induced upregulation of IFN-α and IRF-7, by TSA (100 ng/ml) (B) and MS-275 (5 μM) (C) and SAHA (500 nM) (D). (N = 5; 5 independent experiments with different donors for IAV. N = 3; 3 independent experiments with different donors for HSV-1. Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; (* = p

    Article Snippet: In order to answer this question, PBMC were isolated and pre-treated with TSA or SAHA for 1 hour, then stimulated with HSV-1 for 3 hours, then we utilized BD Phosflow™ assays to assess the phosphorylation status of IRF-7 and NF-κB p65 in the presence of TSA, MS-275, SAHA.

    Techniques: Expressing, Flow Cytometry

    TSA and MS-275 inhibit HSV-1 and IAV-induced IFN-α and TNF-α production in human pDC in a dose-dependent manner. PBMC were incubated with TSA and MS-275 at decreasing concentrations for 1 hour and then stimulated with HSV-1 or IAV for 6 hours. Cells were then processed for intracellular flow cytometry to detect IFN-α and TNF-α production in pDC. Representative histograms showing the differential inhibition of intracellular HSV-1 and IAV-induced IFN-α and TNF-α production in pDC by TSA (100 ng/ml = 330 nM) or DMSO vehicle control (1:1000) (A), and MS-275 (5 μM) or DMSO vehicle control (1:530) (C). Pooled data of dose curve experiments showing a dose-dependent inhibitory effect on HSV-1- (MOI of 1; squares) or IAV (MOI of 2; triangles) -induced IFN-α and TNF-α by TSA (0-100 ng/ml) (C) and MS-275 (0-5 μM) (D) when compared to their respective vehicle controls. (TSA, N=3; MS-275, N=4; 3-4 independent experiments from different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; *p

    Journal: bioRxiv

    Article Title: HDAC inhibitors decrease TLR7/9-mediated human plasmacytoid dendritic cell activation by interfering with IRF-7 and NF-κB signaling

    doi: 10.1101/2020.05.09.085456

    Figure Lengend Snippet: TSA and MS-275 inhibit HSV-1 and IAV-induced IFN-α and TNF-α production in human pDC in a dose-dependent manner. PBMC were incubated with TSA and MS-275 at decreasing concentrations for 1 hour and then stimulated with HSV-1 or IAV for 6 hours. Cells were then processed for intracellular flow cytometry to detect IFN-α and TNF-α production in pDC. Representative histograms showing the differential inhibition of intracellular HSV-1 and IAV-induced IFN-α and TNF-α production in pDC by TSA (100 ng/ml = 330 nM) or DMSO vehicle control (1:1000) (A), and MS-275 (5 μM) or DMSO vehicle control (1:530) (C). Pooled data of dose curve experiments showing a dose-dependent inhibitory effect on HSV-1- (MOI of 1; squares) or IAV (MOI of 2; triangles) -induced IFN-α and TNF-α by TSA (0-100 ng/ml) (C) and MS-275 (0-5 μM) (D) when compared to their respective vehicle controls. (TSA, N=3; MS-275, N=4; 3-4 independent experiments from different donors; Data are expressed as mean ± SEM; Data were analyzed with 1-way ANOVA with Bonferroni’s post test; *p

    Article Snippet: In order to answer this question, PBMC were isolated and pre-treated with TSA or SAHA for 1 hour, then stimulated with HSV-1 for 3 hours, then we utilized BD Phosflow™ assays to assess the phosphorylation status of IRF-7 and NF-κB p65 in the presence of TSA, MS-275, SAHA.

    Techniques: Incubation, Flow Cytometry, Inhibition