Structured Review

Miltenyi Biotec pbmcs
SPM reversed the MGBG inhibitory effects on sOPN and CD16 expression in monocytes. <t>PBMCs</t> were cultured for 3 days with or without treatment. Cells were cultured with 10 μM MGBG and various concentrations of SPM. (A). MGBG significantly decreased sOPN; SPM reversed the MGBG inhibitory effects on sOPN. The average sOPN level of 3 day untreated and 10 μM MGBG treated cells was 30 and 2 ng/ml, respectively. n = 6, means and SEM. (B). MGBG inhibited monocyte CD16 expression; SPM reversed the MGBG inhibitory effects on CD16 expression. Cultured PBMCs were double stained with <t>CD14</t> and CD16 antibodies for flow cytometry analysis. The average CD16 geometric mean in untreated and MGBG treated CD14+ monocytes were measured at 510 and 249, respectively. n = 3, means and SEM. Data was presented as a percentage of inhibition of MGMG treatment only.
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1) Product Images from "Methylglyoxal-bis-guanylhydrazone inhibits osteopontin expression and differentiation in cultured human monocytes"

Article Title: Methylglyoxal-bis-guanylhydrazone inhibits osteopontin expression and differentiation in cultured human monocytes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0192680

SPM reversed the MGBG inhibitory effects on sOPN and CD16 expression in monocytes. PBMCs were cultured for 3 days with or without treatment. Cells were cultured with 10 μM MGBG and various concentrations of SPM. (A). MGBG significantly decreased sOPN; SPM reversed the MGBG inhibitory effects on sOPN. The average sOPN level of 3 day untreated and 10 μM MGBG treated cells was 30 and 2 ng/ml, respectively. n = 6, means and SEM. (B). MGBG inhibited monocyte CD16 expression; SPM reversed the MGBG inhibitory effects on CD16 expression. Cultured PBMCs were double stained with CD14 and CD16 antibodies for flow cytometry analysis. The average CD16 geometric mean in untreated and MGBG treated CD14+ monocytes were measured at 510 and 249, respectively. n = 3, means and SEM. Data was presented as a percentage of inhibition of MGMG treatment only.
Figure Legend Snippet: SPM reversed the MGBG inhibitory effects on sOPN and CD16 expression in monocytes. PBMCs were cultured for 3 days with or without treatment. Cells were cultured with 10 μM MGBG and various concentrations of SPM. (A). MGBG significantly decreased sOPN; SPM reversed the MGBG inhibitory effects on sOPN. The average sOPN level of 3 day untreated and 10 μM MGBG treated cells was 30 and 2 ng/ml, respectively. n = 6, means and SEM. (B). MGBG inhibited monocyte CD16 expression; SPM reversed the MGBG inhibitory effects on CD16 expression. Cultured PBMCs were double stained with CD14 and CD16 antibodies for flow cytometry analysis. The average CD16 geometric mean in untreated and MGBG treated CD14+ monocytes were measured at 510 and 249, respectively. n = 3, means and SEM. Data was presented as a percentage of inhibition of MGMG treatment only.

Techniques Used: Expressing, Cell Culture, Staining, Flow Cytometry, Cytometry, Inhibition

MGBG showed greater inhibition on CD16 expression in monocytes than that in differentiated macrophages. Monocytes were isolated from PBMCs using CD14 microbeads. Cells were cultured for 1, 3, and 6 days with various concentrations of MGBG, and collected for CD16 expression measurement using flow cytometry. MGBG showed greater inhibitory effect on CD 16 expression in 1 and 3 day monocytes than that in differentiated macrophages. The average CD16 geometric mean fluorescence was measured at 108, 448, and 249 for 1, 3, and 6 day untreated cells, respectively. n = 4, means and SEM.
Figure Legend Snippet: MGBG showed greater inhibition on CD16 expression in monocytes than that in differentiated macrophages. Monocytes were isolated from PBMCs using CD14 microbeads. Cells were cultured for 1, 3, and 6 days with various concentrations of MGBG, and collected for CD16 expression measurement using flow cytometry. MGBG showed greater inhibitory effect on CD 16 expression in 1 and 3 day monocytes than that in differentiated macrophages. The average CD16 geometric mean fluorescence was measured at 108, 448, and 249 for 1, 3, and 6 day untreated cells, respectively. n = 4, means and SEM.

Techniques Used: Inhibition, Expressing, Isolation, Cell Culture, Flow Cytometry, Cytometry, Fluorescence

2) Product Images from "Hypoxia Potentiates Glioma-Mediated Immunosuppression"

Article Title: Hypoxia Potentiates Glioma-Mediated Immunosuppression

Journal: PLoS ONE

doi: 10.1371/journal.pone.0016195

Hypoxia enhances the gCSC-mediated immunosuppression on human T cells. A. When healthy donor peripheral blood mononuclear cells (PBMCs) were cultured in the presence of supernatant medium from cultures of each of the gCSCs, T cell proliferation was inhibited, as demonstrated by fluorescence-activated cell sorting (FACS) analysis of CD3+ T cell carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling that measures T cell proliferation. For each of the matched gCSCs under hypoxic conditions, T cell proliferation was further inhibited compared to normoxia. Representative FACS histograms or dot plots are shown on the left, and the summary bar graphs of percentage changes are shown on the right. T cells cultured with neurosphere cell medium served as a control. B. The supernatants from the gCSCs induced an increase in the number of FoxP3 + regulatory T cells in the gated CD4 + T cells, which is further enhanced under conditions of hypoxia. C. The inhibition of IFNγ production in CD3+ T cells was enhanced under hypoxic condition compared to normoxia. * P
Figure Legend Snippet: Hypoxia enhances the gCSC-mediated immunosuppression on human T cells. A. When healthy donor peripheral blood mononuclear cells (PBMCs) were cultured in the presence of supernatant medium from cultures of each of the gCSCs, T cell proliferation was inhibited, as demonstrated by fluorescence-activated cell sorting (FACS) analysis of CD3+ T cell carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling that measures T cell proliferation. For each of the matched gCSCs under hypoxic conditions, T cell proliferation was further inhibited compared to normoxia. Representative FACS histograms or dot plots are shown on the left, and the summary bar graphs of percentage changes are shown on the right. T cells cultured with neurosphere cell medium served as a control. B. The supernatants from the gCSCs induced an increase in the number of FoxP3 + regulatory T cells in the gated CD4 + T cells, which is further enhanced under conditions of hypoxia. C. The inhibition of IFNγ production in CD3+ T cells was enhanced under hypoxic condition compared to normoxia. * P

Techniques Used: Cell Culture, Fluorescence, FACS, Labeling, Inhibition

3) Product Images from "NS1 Specific CD8+ T-Cells with Effector Function and TRBV11 Dominance in a Patient with Parvovirus B19 Associated Inflammatory Cardiomyopathy"

Article Title: NS1 Specific CD8+ T-Cells with Effector Function and TRBV11 Dominance in a Patient with Parvovirus B19 Associated Inflammatory Cardiomyopathy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0002361

TRBV expression of B19V NS1-specific T-cells. Bars indicate the TRBV expression normalized to TRBC in non-selected PBMCs compared with positively and negatively selected GLCP and SALK reactive T-cells which were enriched using the IFNγ secretion assay.
Figure Legend Snippet: TRBV expression of B19V NS1-specific T-cells. Bars indicate the TRBV expression normalized to TRBC in non-selected PBMCs compared with positively and negatively selected GLCP and SALK reactive T-cells which were enriched using the IFNγ secretion assay.

Techniques Used: Expressing

Expression of effector T-cell markers of B19V NS1-specific T-cells. The bars indicate marker expression (normalized to CD3d in non-selected PBMCs) compared with positively and negatively selected GLCP and SALK reactive T-cells enriched using the IFNγ secretion assay. The 3 panels show different target gene expression levels.
Figure Legend Snippet: Expression of effector T-cell markers of B19V NS1-specific T-cells. The bars indicate marker expression (normalized to CD3d in non-selected PBMCs) compared with positively and negatively selected GLCP and SALK reactive T-cells enriched using the IFNγ secretion assay. The 3 panels show different target gene expression levels.

Techniques Used: Expressing, Marker

4) Product Images from "Interleukin (IL)-17A, F and AF in inflammation: a study in collagen-induced arthritis and rheumatoid arthritis"

Article Title: Interleukin (IL)-17A, F and AF in inflammation: a study in collagen-induced arthritis and rheumatoid arthritis

Journal: Clinical and Experimental Immunology

doi: 10.1111/cei.12376

Interleukin (IL)-17A, F and AF responses in purified CD3 + T cells from peripheral blood monuclear cells (PBMCs) or synovial fluid mononuclear cells (SFMCs). Purified CD3 (+) T cells from patients with rheumatoid arthritis (RA) ( n = 9) or osteoarthritis (OA) ( n = 6) were stimulated with phorbol myristate acetate (PMA)/ionomycin for 24 h and IL-17A, IL-17F and IL-17AF were analysed in culture supernatant by enzyme-linked immunosorbent assay (ELISA). ** P
Figure Legend Snippet: Interleukin (IL)-17A, F and AF responses in purified CD3 + T cells from peripheral blood monuclear cells (PBMCs) or synovial fluid mononuclear cells (SFMCs). Purified CD3 (+) T cells from patients with rheumatoid arthritis (RA) ( n = 9) or osteoarthritis (OA) ( n = 6) were stimulated with phorbol myristate acetate (PMA)/ionomycin for 24 h and IL-17A, IL-17F and IL-17AF were analysed in culture supernatant by enzyme-linked immunosorbent assay (ELISA). ** P

Techniques Used: Purification, Enzyme-linked Immunosorbent Assay

5) Product Images from "Minocycline modulates antigen-specific CTL activity through inactivation of mononuclear phagocytes in patients with HTLV-I associated neurologic disease"

Article Title: Minocycline modulates antigen-specific CTL activity through inactivation of mononuclear phagocytes in patients with HTLV-I associated neurologic disease

Journal: Retrovirology

doi: 10.1186/1742-4690-9-16

Activated CD14 + cells in patients with HAM/TSP . (A) TNF-α expression in CD14 + cells of NDs and HAM/TSP patients after culture for 24 hours. The data were obtained from six NDs and six HAM/TSP patients. CD14 + cells expressing TNF-α was significantly elevated in HAM/TSP patients, compared to NDs by Mann-Whitney test (p = 0.0022). The horizontal line represents the mean. (B) Detection of IL-1β in PBMC culture supernatants of NDs and HAM/TSP patients after culture for 24 hours. The data were obtained from five NDs and eight HAM/TSP patients. IL-1β expression in HAM/TSP patients was significantly higher in those cells of NDs by Mann-Whitney test (p = 0.0043). The horizontal line represents the mean. (C) Tax expressions in CD4 + CD25 + T cells (i) and CD4 + CD25 - T cells (ii) cocultured with or without autologous CD14 + cells of ACs (n = 2, opened circle) and patients with HAM/TSP (n = 3, closed circle) for 18 hours.
Figure Legend Snippet: Activated CD14 + cells in patients with HAM/TSP . (A) TNF-α expression in CD14 + cells of NDs and HAM/TSP patients after culture for 24 hours. The data were obtained from six NDs and six HAM/TSP patients. CD14 + cells expressing TNF-α was significantly elevated in HAM/TSP patients, compared to NDs by Mann-Whitney test (p = 0.0022). The horizontal line represents the mean. (B) Detection of IL-1β in PBMC culture supernatants of NDs and HAM/TSP patients after culture for 24 hours. The data were obtained from five NDs and eight HAM/TSP patients. IL-1β expression in HAM/TSP patients was significantly higher in those cells of NDs by Mann-Whitney test (p = 0.0043). The horizontal line represents the mean. (C) Tax expressions in CD4 + CD25 + T cells (i) and CD4 + CD25 - T cells (ii) cocultured with or without autologous CD14 + cells of ACs (n = 2, opened circle) and patients with HAM/TSP (n = 3, closed circle) for 18 hours.

Techniques Used: Expressing, MANN-WHITNEY

Minocycline inhibited TNF-α expression and IL-1β release in patients with HAM/TSP . (A) Dose-dependent inhibitory effects of minocycline on TNF-α expressions in CD14 + cells (closed bar) and CD4 + T cells (opened bar) of HAM/TSP patients (n = 6). The PBMCs were cultured with 0, 5 and 10 μM of minocycline for 24 hours. The frequency of CD14 + cells expressing TNF-α was significantly inhibited at 10 μM of minocycline treatment in HAM/TSP patients (p = 0.0313; Wilcoxon matched-pairs signed rank test). Error bars represent SD. (B) Inhibition of IL-1β release in PBMC culture supernatants of HAM/TSP patients by 10 μM of minocycline after culture for 24 hours (n = 8). The release of IL-1β from these cultured HAM/TSP PBMCs was significantly inhibited by minocycline treatment (p = 0.0078; Wilcoxon matched-pairs signed rank test). (C) Inhibitory effects of minocycline on spontaneous lymphoproliferation in HAM/TSP patients. The PBMCs from two HAM/TSP patients (HAM#1 and #2) were cultured with 0, 2.5, 5 and 10 μM of minocycline, and pulsed with 1 μCi [ 3 H] thymidine for 4 hours at 3 days (closed circle), 4 days (closed square) and 5 days (closed triangle). The average cpm from each well in triplicate was plotted.
Figure Legend Snippet: Minocycline inhibited TNF-α expression and IL-1β release in patients with HAM/TSP . (A) Dose-dependent inhibitory effects of minocycline on TNF-α expressions in CD14 + cells (closed bar) and CD4 + T cells (opened bar) of HAM/TSP patients (n = 6). The PBMCs were cultured with 0, 5 and 10 μM of minocycline for 24 hours. The frequency of CD14 + cells expressing TNF-α was significantly inhibited at 10 μM of minocycline treatment in HAM/TSP patients (p = 0.0313; Wilcoxon matched-pairs signed rank test). Error bars represent SD. (B) Inhibition of IL-1β release in PBMC culture supernatants of HAM/TSP patients by 10 μM of minocycline after culture for 24 hours (n = 8). The release of IL-1β from these cultured HAM/TSP PBMCs was significantly inhibited by minocycline treatment (p = 0.0078; Wilcoxon matched-pairs signed rank test). (C) Inhibitory effects of minocycline on spontaneous lymphoproliferation in HAM/TSP patients. The PBMCs from two HAM/TSP patients (HAM#1 and #2) were cultured with 0, 2.5, 5 and 10 μM of minocycline, and pulsed with 1 μCi [ 3 H] thymidine for 4 hours at 3 days (closed circle), 4 days (closed square) and 5 days (closed triangle). The average cpm from each well in triplicate was plotted.

Techniques Used: Expressing, Cell Culture, Inhibition

6) Product Images from "αβ T-cell receptors from multiple sclerosis brain lesions show MAIT cell–related features"

Article Title: αβ T-cell receptors from multiple sclerosis brain lesions show MAIT cell–related features

Journal: Neurology® Neuroimmunology & Neuroinflammation

doi: 10.1212/NXI.0000000000000107

Expanded Vβ1 chain pairs with MAIT cell–related α chains (A) Cluster of expanded Vβ1 + T cells within multiple sclerosis (MS) lesions. Immunohistochemistry for the expanded and persisting Vβ1 clone (green) in clusters of CD8 + T cells (red) in parenchymal MS lesions. Several such clusters were observed. Only very few scattered CD8 − Vβ1 + T cells could be identified in the brain lesion. Nuclei are visualized with 4',6-diamidino-2-phenylindole (white). Scale bar 20 µm. (B) Sequences of paired T-cell receptor (TCR) α and β chains. Single sorted or laser microdissected Vβ1 + CD8 + T cells from peripheral blood or brain sections were submitted to single-cell TCR PCR to identify Vβ1 chains and all possible matching α chains. The V, n(D)n, and J regions are indicated. Amino acids encoded by n(D)n nucleotides are printed in red. The expanded Vβ1-Jβ2.3 β chain (upper line) was found to pair with 4 different α chains. Three α chains were identified from brain lesions, and 1 α chain was found in blood. The α chains expressing the Jα33 (second line) and Jα16 (fifth line) elements were identified in 7 and 6 independent cells, respectively. All α chains share the Vα7.2 element, and even though they do not share the same Jα element, they all show homologous complementarity determining region 3α regions with a conserved valine (V) followed by a positively charged arginine (R) (with only one clone showing a glutamine [Q]), a negatively charged amino acid (D/E), and a relatively large hydrophilic amino acid. One of the α chains (highlighted in red) is the mucosal-associated invariant T (MAIT) cell canonical TCR Vα7.2-CAXXDSNYQLIW-Jα33 chain with 2 N nucleotide–encoded amino acids between Vα7.2 and Jα33 (here VR). The other clones with Jα16, Jα24.1, and Jα58 chains are atypical for MAIT cells, which usually carry Jα33, Jα20, or Jα12. PBMC = peripheral blood mononuclear cell.
Figure Legend Snippet: Expanded Vβ1 chain pairs with MAIT cell–related α chains (A) Cluster of expanded Vβ1 + T cells within multiple sclerosis (MS) lesions. Immunohistochemistry for the expanded and persisting Vβ1 clone (green) in clusters of CD8 + T cells (red) in parenchymal MS lesions. Several such clusters were observed. Only very few scattered CD8 − Vβ1 + T cells could be identified in the brain lesion. Nuclei are visualized with 4',6-diamidino-2-phenylindole (white). Scale bar 20 µm. (B) Sequences of paired T-cell receptor (TCR) α and β chains. Single sorted or laser microdissected Vβ1 + CD8 + T cells from peripheral blood or brain sections were submitted to single-cell TCR PCR to identify Vβ1 chains and all possible matching α chains. The V, n(D)n, and J regions are indicated. Amino acids encoded by n(D)n nucleotides are printed in red. The expanded Vβ1-Jβ2.3 β chain (upper line) was found to pair with 4 different α chains. Three α chains were identified from brain lesions, and 1 α chain was found in blood. The α chains expressing the Jα33 (second line) and Jα16 (fifth line) elements were identified in 7 and 6 independent cells, respectively. All α chains share the Vα7.2 element, and even though they do not share the same Jα element, they all show homologous complementarity determining region 3α regions with a conserved valine (V) followed by a positively charged arginine (R) (with only one clone showing a glutamine [Q]), a negatively charged amino acid (D/E), and a relatively large hydrophilic amino acid. One of the α chains (highlighted in red) is the mucosal-associated invariant T (MAIT) cell canonical TCR Vα7.2-CAXXDSNYQLIW-Jα33 chain with 2 N nucleotide–encoded amino acids between Vα7.2 and Jα33 (here VR). The other clones with Jα16, Jα24.1, and Jα58 chains are atypical for MAIT cells, which usually carry Jα33, Jα20, or Jα12. PBMC = peripheral blood mononuclear cell.

Techniques Used: Mass Spectrometry, Immunohistochemistry, Polymerase Chain Reaction, Expressing, Clone Assay

Vα7.2 TCR chain repertoire Analysis of the T-cell receptor (TCR) Vα7.2 repertoire of patient A by pyrosequencing shows oligoclonal T-cell expansions in different samples. Each pie chart represents the total number of nucleotide sequences found in the Vα7.2 repertoire of a certain compartment and each sector represents one distinct T-cell clone. We compared (A) samples from CNS tissue from the biopsy taken in 1996, (B) Vα7.2 + CD161 + mucosal-associated invariant T (MAIT) cells, (C) Vα7.2 + CD161 − cells, (D) CD8 + cells, and (E) CD4 + T cells from peripheral blood taken in 2013 and 2014. For each population we list the designation of the Jα elements and their relative percentage. Clones carrying the MAIT canonical TCR α chain (CAXXDSNYQLIW) are marked in bright blue, and clones containing the noncanonical α chains characterized by Jα12 (CAXXDSSYKLIF) and Jα20 (CAVXXDYKLSF) are marked in dark blue and light blue, respectively. (F) Percentages of identical complementarity determining region 3α amino acid sequences within the TCR Vα7.2 + repertoire between the different samples defined above (A–E). The numbers are percentages indicating how often a particular sequence detected in one sample was also found in another sample. The greatest overlap was between Vα7.2 + CD161 + and CD8 + T cells from peripheral blood, but there was also significant overlap between the CNS sample and the Vα7.2 + CD161 + sample from 2013 to 2014, as highlighted by the red and yellow colors. PBMC = peripheral blood mononuclear cell.
Figure Legend Snippet: Vα7.2 TCR chain repertoire Analysis of the T-cell receptor (TCR) Vα7.2 repertoire of patient A by pyrosequencing shows oligoclonal T-cell expansions in different samples. Each pie chart represents the total number of nucleotide sequences found in the Vα7.2 repertoire of a certain compartment and each sector represents one distinct T-cell clone. We compared (A) samples from CNS tissue from the biopsy taken in 1996, (B) Vα7.2 + CD161 + mucosal-associated invariant T (MAIT) cells, (C) Vα7.2 + CD161 − cells, (D) CD8 + cells, and (E) CD4 + T cells from peripheral blood taken in 2013 and 2014. For each population we list the designation of the Jα elements and their relative percentage. Clones carrying the MAIT canonical TCR α chain (CAXXDSNYQLIW) are marked in bright blue, and clones containing the noncanonical α chains characterized by Jα12 (CAXXDSSYKLIF) and Jα20 (CAVXXDYKLSF) are marked in dark blue and light blue, respectively. (F) Percentages of identical complementarity determining region 3α amino acid sequences within the TCR Vα7.2 + repertoire between the different samples defined above (A–E). The numbers are percentages indicating how often a particular sequence detected in one sample was also found in another sample. The greatest overlap was between Vα7.2 + CD161 + and CD8 + T cells from peripheral blood, but there was also significant overlap between the CNS sample and the Vα7.2 + CD161 + sample from 2013 to 2014, as highlighted by the red and yellow colors. PBMC = peripheral blood mononuclear cell.

Techniques Used: Clone Assay, Sequencing

7) Product Images from "αβ T-cell receptors from multiple sclerosis brain lesions show MAIT cell–related features"

Article Title: αβ T-cell receptors from multiple sclerosis brain lesions show MAIT cell–related features

Journal: Neurology® Neuroimmunology & Neuroinflammation

doi: 10.1212/NXI.0000000000000107

Expanded Vβ1 chain pairs with MAIT cell–related α chains (A) Cluster of expanded Vβ1 + T cells within multiple sclerosis (MS) lesions. Immunohistochemistry for the expanded and persisting Vβ1 clone (green) in clusters of CD8 + T cells (red) in parenchymal MS lesions. Several such clusters were observed. Only very few scattered CD8 − Vβ1 + T cells could be identified in the brain lesion. Nuclei are visualized with 4',6-diamidino-2-phenylindole (white). Scale bar 20 µm. (B) Sequences of paired T-cell receptor (TCR) α and β chains. Single sorted or laser microdissected Vβ1 + CD8 + T cells from peripheral blood or brain sections were submitted to single-cell TCR PCR to identify Vβ1 chains and all possible matching α chains. The V, n(D)n, and J regions are indicated. Amino acids encoded by n(D)n nucleotides are printed in red. The expanded Vβ1-Jβ2.3 β chain (upper line) was found to pair with 4 different α chains. Three α chains were identified from brain lesions, and 1 α chain was found in blood. The α chains expressing the Jα33 (second line) and Jα16 (fifth line) elements were identified in 7 and 6 independent cells, respectively. All α chains share the Vα7.2 element, and even though they do not share the same Jα element, they all show homologous complementarity determining region 3α regions with a conserved valine (V) followed by a positively charged arginine (R) (with only one clone showing a glutamine [Q]), a negatively charged amino acid (D/E), and a relatively large hydrophilic amino acid. One of the α chains (highlighted in red) is the mucosal-associated invariant T (MAIT) cell canonical TCR Vα7.2-CAXXDSNYQLIW-Jα33 chain with 2 N nucleotide–encoded amino acids between Vα7.2 and Jα33 (here VR). The other clones with Jα16, Jα24.1, and Jα58 chains are atypical for MAIT cells, which usually carry Jα33, Jα20, or Jα12. PBMC = peripheral blood mononuclear cell.
Figure Legend Snippet: Expanded Vβ1 chain pairs with MAIT cell–related α chains (A) Cluster of expanded Vβ1 + T cells within multiple sclerosis (MS) lesions. Immunohistochemistry for the expanded and persisting Vβ1 clone (green) in clusters of CD8 + T cells (red) in parenchymal MS lesions. Several such clusters were observed. Only very few scattered CD8 − Vβ1 + T cells could be identified in the brain lesion. Nuclei are visualized with 4',6-diamidino-2-phenylindole (white). Scale bar 20 µm. (B) Sequences of paired T-cell receptor (TCR) α and β chains. Single sorted or laser microdissected Vβ1 + CD8 + T cells from peripheral blood or brain sections were submitted to single-cell TCR PCR to identify Vβ1 chains and all possible matching α chains. The V, n(D)n, and J regions are indicated. Amino acids encoded by n(D)n nucleotides are printed in red. The expanded Vβ1-Jβ2.3 β chain (upper line) was found to pair with 4 different α chains. Three α chains were identified from brain lesions, and 1 α chain was found in blood. The α chains expressing the Jα33 (second line) and Jα16 (fifth line) elements were identified in 7 and 6 independent cells, respectively. All α chains share the Vα7.2 element, and even though they do not share the same Jα element, they all show homologous complementarity determining region 3α regions with a conserved valine (V) followed by a positively charged arginine (R) (with only one clone showing a glutamine [Q]), a negatively charged amino acid (D/E), and a relatively large hydrophilic amino acid. One of the α chains (highlighted in red) is the mucosal-associated invariant T (MAIT) cell canonical TCR Vα7.2-CAXXDSNYQLIW-Jα33 chain with 2 N nucleotide–encoded amino acids between Vα7.2 and Jα33 (here VR). The other clones with Jα16, Jα24.1, and Jα58 chains are atypical for MAIT cells, which usually carry Jα33, Jα20, or Jα12. PBMC = peripheral blood mononuclear cell.

Techniques Used: Mass Spectrometry, Immunohistochemistry, Polymerase Chain Reaction, Expressing, Clone Assay

Vα7.2 TCR chain repertoire Analysis of the T-cell receptor (TCR) Vα7.2 repertoire of patient A by pyrosequencing shows oligoclonal T-cell expansions in different samples. Each pie chart represents the total number of nucleotide sequences found in the Vα7.2 repertoire of a certain compartment and each sector represents one distinct T-cell clone. We compared (A) samples from CNS tissue from the biopsy taken in 1996, (B) Vα7.2 + CD161 + mucosal-associated invariant T (MAIT) cells, (C) Vα7.2 + CD161 − cells, (D) CD8 + cells, and (E) CD4 + T cells from peripheral blood taken in 2013 and 2014. For each population we list the designation of the Jα elements and their relative percentage. Clones carrying the MAIT canonical TCR α chain (CAXXDSNYQLIW) are marked in bright blue, and clones containing the noncanonical α chains characterized by Jα12 (CAXXDSSYKLIF) and Jα20 (CAVXXDYKLSF) are marked in dark blue and light blue, respectively. (F) Percentages of identical complementarity determining region 3α amino acid sequences within the TCR Vα7.2 + repertoire between the different samples defined above (A–E). The numbers are percentages indicating how often a particular sequence detected in one sample was also found in another sample. The greatest overlap was between Vα7.2 + CD161 + and CD8 + T cells from peripheral blood, but there was also significant overlap between the CNS sample and the Vα7.2 + CD161 + sample from 2013 to 2014, as highlighted by the red and yellow colors. PBMC = peripheral blood mononuclear cell.
Figure Legend Snippet: Vα7.2 TCR chain repertoire Analysis of the T-cell receptor (TCR) Vα7.2 repertoire of patient A by pyrosequencing shows oligoclonal T-cell expansions in different samples. Each pie chart represents the total number of nucleotide sequences found in the Vα7.2 repertoire of a certain compartment and each sector represents one distinct T-cell clone. We compared (A) samples from CNS tissue from the biopsy taken in 1996, (B) Vα7.2 + CD161 + mucosal-associated invariant T (MAIT) cells, (C) Vα7.2 + CD161 − cells, (D) CD8 + cells, and (E) CD4 + T cells from peripheral blood taken in 2013 and 2014. For each population we list the designation of the Jα elements and their relative percentage. Clones carrying the MAIT canonical TCR α chain (CAXXDSNYQLIW) are marked in bright blue, and clones containing the noncanonical α chains characterized by Jα12 (CAXXDSSYKLIF) and Jα20 (CAVXXDYKLSF) are marked in dark blue and light blue, respectively. (F) Percentages of identical complementarity determining region 3α amino acid sequences within the TCR Vα7.2 + repertoire between the different samples defined above (A–E). The numbers are percentages indicating how often a particular sequence detected in one sample was also found in another sample. The greatest overlap was between Vα7.2 + CD161 + and CD8 + T cells from peripheral blood, but there was also significant overlap between the CNS sample and the Vα7.2 + CD161 + sample from 2013 to 2014, as highlighted by the red and yellow colors. PBMC = peripheral blood mononuclear cell.

Techniques Used: Clone Assay, Sequencing

8) Product Images from "Antigen responsive CD4+ T cell clones contribute to the HIV-1 latent reservoir"

Article Title: Antigen responsive CD4+ T cell clones contribute to the HIV-1 latent reservoir

Journal: bioRxiv

doi: 10.1101/2020.01.10.902155

(a) Experimental overview. PBMCs were depleted of CD8 + T cells and rested for 3h before stimulation for 18h with peptide pools. Cells were then purified based on expression of activation induced markers (AIM) (CD69, PD-L1 and 4-1BB, Supplemental Figure 1 ). DNA was isolated from sorted cells, Q4PCR was performed, and sequenced viruses were assembled and analyzed. (b) Representative example of cell purification by AIM after stimulation with CMV-pp65, HIV-gag, CEFT (from 4/8 donors) or SEB by gating on CD69-positive cells followed by gating on PD-L1 positive or 4-1BB positive cells. Control CD4 + T cells cultured with MOG were purified on the basis of CD4 expression alone. Each AIM assay staining was performed twice on each donor. Numbers represent the percentage of total CD4 + T cells within each gate. (c) Frequency of AIM+ cells across all donors. Cells were processed and sorted as in (b).
Figure Legend Snippet: (a) Experimental overview. PBMCs were depleted of CD8 + T cells and rested for 3h before stimulation for 18h with peptide pools. Cells were then purified based on expression of activation induced markers (AIM) (CD69, PD-L1 and 4-1BB, Supplemental Figure 1 ). DNA was isolated from sorted cells, Q4PCR was performed, and sequenced viruses were assembled and analyzed. (b) Representative example of cell purification by AIM after stimulation with CMV-pp65, HIV-gag, CEFT (from 4/8 donors) or SEB by gating on CD69-positive cells followed by gating on PD-L1 positive or 4-1BB positive cells. Control CD4 + T cells cultured with MOG were purified on the basis of CD4 expression alone. Each AIM assay staining was performed twice on each donor. Numbers represent the percentage of total CD4 + T cells within each gate. (c) Frequency of AIM+ cells across all donors. Cells were processed and sorted as in (b).

Techniques Used: Purification, Expressing, Activation Assay, Isolation, Cell Culture, Staining

9) Product Images from "Additive anti-inflammatory effects of corticosteroids and phosphodiesterase-4 inhibitors in COPD CD8 cells"

Article Title: Additive anti-inflammatory effects of corticosteroids and phosphodiesterase-4 inhibitors in COPD CD8 cells

Journal: Respiratory Research

doi: 10.1186/s12931-016-0325-8

The effect of PDE4 inhibitors with dexamethasone on IL-2 release from peripheral blood CD8 cells and PBMCs. Peripheral blood CD8 cells and PBMCs from COPD ( n = 13, a b ), Smoker ( n = 8, c d ) and Healthy non-smokers (HNS, n = 7 e f ) were pre-treated for 1 h with various concentrations of dexamethasone (Dex) alone (О) or in combination with GSK256066 (■), Roflumilast (♦) or Forskolin (▲). The effect of GSK256066, roflumilast and forskolin alone is represented as log[Dex] = 0. Cells were stimulated for 24 h with anti-CD2/3/28 beads. Supernatants were harvested and Interleukin 2 (IL-2) was measured by ELISA. Data presented as mean ± SE
Figure Legend Snippet: The effect of PDE4 inhibitors with dexamethasone on IL-2 release from peripheral blood CD8 cells and PBMCs. Peripheral blood CD8 cells and PBMCs from COPD ( n = 13, a b ), Smoker ( n = 8, c d ) and Healthy non-smokers (HNS, n = 7 e f ) were pre-treated for 1 h with various concentrations of dexamethasone (Dex) alone (О) or in combination with GSK256066 (■), Roflumilast (♦) or Forskolin (▲). The effect of GSK256066, roflumilast and forskolin alone is represented as log[Dex] = 0. Cells were stimulated for 24 h with anti-CD2/3/28 beads. Supernatants were harvested and Interleukin 2 (IL-2) was measured by ELISA. Data presented as mean ± SE

Techniques Used: Enzyme-linked Immunosorbent Assay

The effect of PDE4 inhibitors with dexamethasone on IFNγ release from peripheral blood CD8 cells and PBMCs. Peripheral blood CD8 cells and PBMCs from COPD ( n = 13, a b ), Smoker ( n = 8 c d ) and Healthy non-smokers (HNS, n = 7, e f ) were pre-treated for 1 h with various concentrations of dexamethasone (Dex) alone (О) or in combination with GSK256066 (■), Roflumilast (♦) or Forskolin (▲). The effect of GSK256066, roflumilast and forskolin alone is represented as log[Dex]M = 0. Cells were stimulated for 24 h with anti-CD2/3/28 beads. Supernatants were harvested and Interferon gamma (IFNγ) was measured by ELISA. Data presented as mean ± SE
Figure Legend Snippet: The effect of PDE4 inhibitors with dexamethasone on IFNγ release from peripheral blood CD8 cells and PBMCs. Peripheral blood CD8 cells and PBMCs from COPD ( n = 13, a b ), Smoker ( n = 8 c d ) and Healthy non-smokers (HNS, n = 7, e f ) were pre-treated for 1 h with various concentrations of dexamethasone (Dex) alone (О) or in combination with GSK256066 (■), Roflumilast (♦) or Forskolin (▲). The effect of GSK256066, roflumilast and forskolin alone is represented as log[Dex]M = 0. Cells were stimulated for 24 h with anti-CD2/3/28 beads. Supernatants were harvested and Interferon gamma (IFNγ) was measured by ELISA. Data presented as mean ± SE

Techniques Used: Enzyme-linked Immunosorbent Assay

10) Product Images from "Two Genetic Variations in the IRF8 region are associated with Behçet’s disease in Han Chinese"

Article Title: Two Genetic Variations in the IRF8 region are associated with Behçet’s disease in Han Chinese

Journal: Scientific Reports

doi: 10.1038/srep19651

The influence of rs17445836 genotypes on cytokine production. The production of IFN-γ ( a ), IL-10 ( b ), IL-17 ( c ) in PBMCs obtained from healthy genotype controls. PBMCs were treated with anti-CD3/28 antibodies. Data show the mean ± SD. Pc: Bonferroni corrected p value, multiplied by 3.
Figure Legend Snippet: The influence of rs17445836 genotypes on cytokine production. The production of IFN-γ ( a ), IL-10 ( b ), IL-17 ( c ) in PBMCs obtained from healthy genotype controls. PBMCs were treated with anti-CD3/28 antibodies. Data show the mean ± SD. Pc: Bonferroni corrected p value, multiplied by 3.

Techniques Used:

The influence of rs17445836 and rs11642873 on the relative expression of IRF8. The expression of IRF8 in PBMCs treated with anti-CD3/28 antibodies. PBMCs were obtained from healthy individuals with diverse genotypes of rs17445836 ( a ) and rs11642873 ( b ). Data show the mean ± SD. Pc: Bonferroni corrected p value, multiplied by 3.
Figure Legend Snippet: The influence of rs17445836 and rs11642873 on the relative expression of IRF8. The expression of IRF8 in PBMCs treated with anti-CD3/28 antibodies. PBMCs were obtained from healthy individuals with diverse genotypes of rs17445836 ( a ) and rs11642873 ( b ). Data show the mean ± SD. Pc: Bonferroni corrected p value, multiplied by 3.

Techniques Used: Expressing

The influence of rs11642873 genotypes on cytokine production. The production of IFN-γ ( a ), IL-10 ( b ), IL-17 ( c ) in PBMCs treated with anti-CD3/28 antibodies. PBMCs were obtained from healthy genotyped controls. Data show the mean ± SD. Pc: Bonferroni corrected p value, multiplied by 3.
Figure Legend Snippet: The influence of rs11642873 genotypes on cytokine production. The production of IFN-γ ( a ), IL-10 ( b ), IL-17 ( c ) in PBMCs treated with anti-CD3/28 antibodies. PBMCs were obtained from healthy genotyped controls. Data show the mean ± SD. Pc: Bonferroni corrected p value, multiplied by 3.

Techniques Used:

11) Product Images from "Classical natural ovine scrapie prions detected in practical volumes of blood by lamb and transgenic mouse bioassays"

Article Title: Classical natural ovine scrapie prions detected in practical volumes of blood by lamb and transgenic mouse bioassays

Journal: Journal of Veterinary Science

doi: 10.4142/jvs.2015.16.2.179

Representative Western immunoblots for detecting PrP res in Tg338 mice inoculated with PBMCs and B lymphocytes. (A) PrP res -specific bands were detected for the brain homogenates of preclinical scrapie donor sheep 4125 (lane 1) along with brain homogenates of Tg338 mice inoculated with PBMCs (lanes 2 and 3), CD72 + B lymphocytes (lanes 4 and 5), and CD21 + B lymphocytes (lanes 6 and 7). These bands were not observed for the uninoculated control mouse (lane 8). (B) PrP res bands were also detected for the brain homogenates of clinical scrapie donor sheep 4124 (lane 1) as well as spleen homogenates of Tg338 mice inoculated with sIgM + B lymphocytes (lanes 2~4) and CD21 + B lymphocytes (lanes 5~7). These bands were not observed for the uninoculated control mouse (lane 8). mAb F99/97.6.1 (2.5 µg/mL) was used to detect PrP res .
Figure Legend Snippet: Representative Western immunoblots for detecting PrP res in Tg338 mice inoculated with PBMCs and B lymphocytes. (A) PrP res -specific bands were detected for the brain homogenates of preclinical scrapie donor sheep 4125 (lane 1) along with brain homogenates of Tg338 mice inoculated with PBMCs (lanes 2 and 3), CD72 + B lymphocytes (lanes 4 and 5), and CD21 + B lymphocytes (lanes 6 and 7). These bands were not observed for the uninoculated control mouse (lane 8). (B) PrP res bands were also detected for the brain homogenates of clinical scrapie donor sheep 4124 (lane 1) as well as spleen homogenates of Tg338 mice inoculated with sIgM + B lymphocytes (lanes 2~4) and CD21 + B lymphocytes (lanes 5~7). These bands were not observed for the uninoculated control mouse (lane 8). mAb F99/97.6.1 (2.5 µg/mL) was used to detect PrP res .

Techniques Used: Western Blot, Mouse Assay

Detection of PrP Sc -specific immunolabeling in the lymphoid tissues of inoculated sheep. PrP Sc (dark red) was visible in the follicles of retropharyngeal lymph nodes collected during necropsy from sheep inoculated with PBMCs (A) or B lymphocytes recovered from 10 (B) and 5 mL (C) of blood. PrP Sc immunolabeling was not detected in uninoculated control sheep (D). Immunohistochemistry (IHC) was performed using a mixture of mAbs F99/97.6.1. and F89/160.1.5. (2.5 µg/mL each) with 3-amino-9-ethylcarbazole (AEC) chromagen. Scale bar = 100 µm
Figure Legend Snippet: Detection of PrP Sc -specific immunolabeling in the lymphoid tissues of inoculated sheep. PrP Sc (dark red) was visible in the follicles of retropharyngeal lymph nodes collected during necropsy from sheep inoculated with PBMCs (A) or B lymphocytes recovered from 10 (B) and 5 mL (C) of blood. PrP Sc immunolabeling was not detected in uninoculated control sheep (D). Immunohistochemistry (IHC) was performed using a mixture of mAbs F99/97.6.1. and F89/160.1.5. (2.5 µg/mL each) with 3-amino-9-ethylcarbazole (AEC) chromagen. Scale bar = 100 µm

Techniques Used: Immunolabeling, Immunohistochemistry

12) Product Images from "Rab27a controls HIV-1 assembly by regulating plasma membrane levels of phosphatidylinositol 4,5-bisphosphate"

Article Title: Rab27a controls HIV-1 assembly by regulating plasma membrane levels of phosphatidylinositol 4,5-bisphosphate

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201409082

Rab27a controls Pr55 Gag recruitment to cell membranes in CD4 + T cells and macrophages. (A) 3D deconvolution fluorescence microscopy of HIV-1–infected PBMCs from a healthy control or a GS patient stained at day 4 p.i. with anti-p24 (green) and anti-CD81 antibodies. Shown is a 3D maximum intensity projection of 10 optical sections acquired at 0.2-µm intervals. (B) Quantitation of PM versus cytosolic distribution of Gag was evaluated by blinded observers on a per-cell basis, in 100 cells of each condition. Data are expressed as percentages of cells in each category. (C) LSCM of HIV-1–infected control or Rab27a-silenced MDMs stained at day 5 p.i. with anti-p24 (green) and anti-CD81 antibodies. (D) Quantification of Gag distribution was performed as described in B. ***, P
Figure Legend Snippet: Rab27a controls Pr55 Gag recruitment to cell membranes in CD4 + T cells and macrophages. (A) 3D deconvolution fluorescence microscopy of HIV-1–infected PBMCs from a healthy control or a GS patient stained at day 4 p.i. with anti-p24 (green) and anti-CD81 antibodies. Shown is a 3D maximum intensity projection of 10 optical sections acquired at 0.2-µm intervals. (B) Quantitation of PM versus cytosolic distribution of Gag was evaluated by blinded observers on a per-cell basis, in 100 cells of each condition. Data are expressed as percentages of cells in each category. (C) LSCM of HIV-1–infected control or Rab27a-silenced MDMs stained at day 5 p.i. with anti-p24 (green) and anti-CD81 antibodies. (D) Quantification of Gag distribution was performed as described in B. ***, P

Techniques Used: Fluorescence, Microscopy, Infection, Staining, Quantitation Assay

Rab27a is required for HIV replication in CD4 + T lymphocytes and macrophages. (A–D) Jurkat cells were transduced with lentivirus encoding different Rab27a shRNA sequences (A and B; open bars), or a control scrambled shRNA (black bars). (A) Rab27a mRNA levels were determined by qPCR. Means ± SD of a representative experiment ( n = 3) performed in triplicates is shown. (B) Cells (3 × 10 4 /0.1 ml) were infected with a VSV-G–pseudotyped HIV (20 ng p24/ml), and p24 production was evaluated in cell supernatants at day 5 p.i. Means ± SD of two independent experiments expressed as percentages of p24 production of control cells are shown. (C) Immunoblot of Rab27a protein levels. Actin was used as a loading control. (D) Cell viability was determined at day 7 after puromycin selection by annexin V staining and propidium iodide exclusion followed by FACS analysis. Dot plots from a representative experiment ( n = 4) are shown. Percentage of death cells (annexin V + /propidium iodide + ) is indicated. (E–G) Control (closed circles) and Rab27a-silenced Jurkat cells (open circles; 3 × 10 4 /0.1 ml) were infected with HIV (IIIb strain; 50 ng p24/ml). The production of p24 (E) and the percentages of p24-positive cells (F and G) were determined by ELISA and FACS analysis, respectively. Representative dot plots (F) and the percentages of infected cells (G) from a representative experiment ( n = 6) are shown. (H) Virus release assay. Cells were spinoculated with a high MOI of VSV-G–pseudotyped HIV strain. 48 h later, the amounts of cell-associated and released virus were analyzed by immunoblotting. (I and J) Control and Rab27a-silenced PBMCs (I) or PBMCs from a GS patient and an age-matched control (J) were infected with a VSV-G–pseudotyped HIV-1 (200 and 50 ng/ml p24, respectively). The production of p24 was evaluated at different days p.i. Results from a representative experiment ( n = 3 and n = 2) performed in triplicates are expressed as the means ± SD. (K) Quantification of p24 antigen in cell supernatants from control (closed triangles) or Rab27a-silenced (open triangles) MDMs (5 × 10 4 /0.1 ml) infected with HIV-1(BaL strain; 50 ng p24/ml). Kinetics of p24 production from a representative experiment are shown in the left graph. p24 production by cells from seven different blood donors at day 5 p.i. is shown in the right graph. Asterisks indicate statistically significant differences from the control: **, P
Figure Legend Snippet: Rab27a is required for HIV replication in CD4 + T lymphocytes and macrophages. (A–D) Jurkat cells were transduced with lentivirus encoding different Rab27a shRNA sequences (A and B; open bars), or a control scrambled shRNA (black bars). (A) Rab27a mRNA levels were determined by qPCR. Means ± SD of a representative experiment ( n = 3) performed in triplicates is shown. (B) Cells (3 × 10 4 /0.1 ml) were infected with a VSV-G–pseudotyped HIV (20 ng p24/ml), and p24 production was evaluated in cell supernatants at day 5 p.i. Means ± SD of two independent experiments expressed as percentages of p24 production of control cells are shown. (C) Immunoblot of Rab27a protein levels. Actin was used as a loading control. (D) Cell viability was determined at day 7 after puromycin selection by annexin V staining and propidium iodide exclusion followed by FACS analysis. Dot plots from a representative experiment ( n = 4) are shown. Percentage of death cells (annexin V + /propidium iodide + ) is indicated. (E–G) Control (closed circles) and Rab27a-silenced Jurkat cells (open circles; 3 × 10 4 /0.1 ml) were infected with HIV (IIIb strain; 50 ng p24/ml). The production of p24 (E) and the percentages of p24-positive cells (F and G) were determined by ELISA and FACS analysis, respectively. Representative dot plots (F) and the percentages of infected cells (G) from a representative experiment ( n = 6) are shown. (H) Virus release assay. Cells were spinoculated with a high MOI of VSV-G–pseudotyped HIV strain. 48 h later, the amounts of cell-associated and released virus were analyzed by immunoblotting. (I and J) Control and Rab27a-silenced PBMCs (I) or PBMCs from a GS patient and an age-matched control (J) were infected with a VSV-G–pseudotyped HIV-1 (200 and 50 ng/ml p24, respectively). The production of p24 was evaluated at different days p.i. Results from a representative experiment ( n = 3 and n = 2) performed in triplicates are expressed as the means ± SD. (K) Quantification of p24 antigen in cell supernatants from control (closed triangles) or Rab27a-silenced (open triangles) MDMs (5 × 10 4 /0.1 ml) infected with HIV-1(BaL strain; 50 ng p24/ml). Kinetics of p24 production from a representative experiment are shown in the left graph. p24 production by cells from seven different blood donors at day 5 p.i. is shown in the right graph. Asterisks indicate statistically significant differences from the control: **, P

Techniques Used: Transduction, shRNA, Real-time Polymerase Chain Reaction, Infection, Selection, Staining, FACS, Enzyme-linked Immunosorbent Assay, Release Assay

13) Product Images from "New insights into the heterogeneity of Th17 subsets contributing to HIV-1 persistence during antiretroviral therapy"

Article Title: New insights into the heterogeneity of Th17 subsets contributing to HIV-1 persistence during antiretroviral therapy

Journal: Retrovirology

doi: 10.1186/s12977-016-0293-6

CCR6 + DN and CCR6 + DP subsets are permissive to HIV infection in vitro. a PBMCs from healthy individuals were stained with a cocktail of fluorochrome-conjugated CD3, CD4, CD45RA, CCR4, CXCR3, CCR6, and CCR5 or CXCR4 Abs. The gating strategy for the identification of distinct CCR6 + and CCR6 − T-cell subsets was designed as in Fig. 1 a. The frequency of cells expressing CCR5 ( left panel ) and CXCR4 ( right panel ) was analyzed within the Th17, Th1Th17, CCR6 + DN and CCR6 + DP subsets. Paired t-Test p -values are indicated on the figures. Horizontal bars indicate median values. b Memory CCR6 + subsets from three HIV-uninfected subjects were sorted and stimulated via CD3/CD28 for 4 days, as in Fig. 1 e. Cells were exposed to a highly infectious R5 strain HIV-ADA8. Levels of HIV-p24 were quantified by ELISA in cell supernatants at day 3 posy-infection
Figure Legend Snippet: CCR6 + DN and CCR6 + DP subsets are permissive to HIV infection in vitro. a PBMCs from healthy individuals were stained with a cocktail of fluorochrome-conjugated CD3, CD4, CD45RA, CCR4, CXCR3, CCR6, and CCR5 or CXCR4 Abs. The gating strategy for the identification of distinct CCR6 + and CCR6 − T-cell subsets was designed as in Fig. 1 a. The frequency of cells expressing CCR5 ( left panel ) and CXCR4 ( right panel ) was analyzed within the Th17, Th1Th17, CCR6 + DN and CCR6 + DP subsets. Paired t-Test p -values are indicated on the figures. Horizontal bars indicate median values. b Memory CCR6 + subsets from three HIV-uninfected subjects were sorted and stimulated via CD3/CD28 for 4 days, as in Fig. 1 e. Cells were exposed to a highly infectious R5 strain HIV-ADA8. Levels of HIV-p24 were quantified by ELISA in cell supernatants at day 3 posy-infection

Techniques Used: Infection, In Vitro, Staining, Expressing, Enzyme-linked Immunosorbent Assay

CCR6 + DN distinguish from the other Th17-subsets by superior frequency/counts in CI on ART individuals. a PBMCs from CI on ART individuals (n = 20; Additional file 8 : Table S2) were stained as in Fig. 1 . Shown is the relative frequency of the four CCR6 + subsets in CI on ART individuals. Paired t-Test p -values are indicated on the figures . Horizontal bars indicate median values. b Shown are median frequencies of CM, TM, and EM within each CCR6 + subsets from CI on ART (n = 10). Shown is the frequency ( c ) and counts ( d ) of the four CCR6 + subsets in CI on ART versus uninfected individuals. Cell counts were calculated taking into account their frequency within the total CD4 + T-cell fraction. e The dynamics of CCR6 + subset counts were investigated longitudinally in n = 5 HIV-infected individuals from the Montreal HIV Primary infection cohort (Additional file 8 : Table S3), in relationship with plasma viral load, before and after ART initiation ( grey ). f Shown are statistical analysis for differences in cell counts between the four CCR6 + subsets (n = 5 HIV-infected individuals) using Friedman test and the post-test Dunn’s multiple comparison and Wilcoxon t test
Figure Legend Snippet: CCR6 + DN distinguish from the other Th17-subsets by superior frequency/counts in CI on ART individuals. a PBMCs from CI on ART individuals (n = 20; Additional file 8 : Table S2) were stained as in Fig. 1 . Shown is the relative frequency of the four CCR6 + subsets in CI on ART individuals. Paired t-Test p -values are indicated on the figures . Horizontal bars indicate median values. b Shown are median frequencies of CM, TM, and EM within each CCR6 + subsets from CI on ART (n = 10). Shown is the frequency ( c ) and counts ( d ) of the four CCR6 + subsets in CI on ART versus uninfected individuals. Cell counts were calculated taking into account their frequency within the total CD4 + T-cell fraction. e The dynamics of CCR6 + subset counts were investigated longitudinally in n = 5 HIV-infected individuals from the Montreal HIV Primary infection cohort (Additional file 8 : Table S3), in relationship with plasma viral load, before and after ART initiation ( grey ). f Shown are statistical analysis for differences in cell counts between the four CCR6 + subsets (n = 5 HIV-infected individuals) using Friedman test and the post-test Dunn’s multiple comparison and Wilcoxon t test

Techniques Used: Staining, Infection

CCR6 + DN are predominant in lymph nodes of HIV-infected individuals receiving ART. a , b Matched PBMCs and inguinal lymph node cells from three CI on ART individuals (CI 36, CI 37, CI 38; Additional file 8 : S2 Table) were stained with a cocktail of fluorochrome-conjugated CD3, CD4, CD45RA, CCR4, CXCR3, CCR6, and CCR7 Abs. A viability staining was used to exclude dead cells. Viable memory CD4 + T-cells (CD3 + CD4 + CD45RA − ) expressing CCR6 were analyzed for their differential expression of CCR4 and CXCR3. The four CCR6 + subsets including Th17, CCR6 + DP, CCR6 + DN, and Th1Th17 were identified in both PBMCs and cells from lymph nodes. a Shown is the phenotype of PBMCs ( upper panels ) and lymph node cells ( lower panels ) in one representative donor. b Shown are statistical analysis of the frequency of CCR6 + subsets in the lymph node (n = 3). Paired t-Test p -values are indicated on the figures
Figure Legend Snippet: CCR6 + DN are predominant in lymph nodes of HIV-infected individuals receiving ART. a , b Matched PBMCs and inguinal lymph node cells from three CI on ART individuals (CI 36, CI 37, CI 38; Additional file 8 : S2 Table) were stained with a cocktail of fluorochrome-conjugated CD3, CD4, CD45RA, CCR4, CXCR3, CCR6, and CCR7 Abs. A viability staining was used to exclude dead cells. Viable memory CD4 + T-cells (CD3 + CD4 + CD45RA − ) expressing CCR6 were analyzed for their differential expression of CCR4 and CXCR3. The four CCR6 + subsets including Th17, CCR6 + DP, CCR6 + DN, and Th1Th17 were identified in both PBMCs and cells from lymph nodes. a Shown is the phenotype of PBMCs ( upper panels ) and lymph node cells ( lower panels ) in one representative donor. b Shown are statistical analysis of the frequency of CCR6 + subsets in the lymph node (n = 3). Paired t-Test p -values are indicated on the figures

Techniques Used: Infection, Staining, Expressing

14) Product Images from "Cytotoxic T lymphocyte-associated antigen-4-Ig (CTLA-4-Ig) suppresses Staphylococcus aureus-induced CD80, CD86, and pro-inflammatory cytokine expression in human B cells"

Article Title: Cytotoxic T lymphocyte-associated antigen-4-Ig (CTLA-4-Ig) suppresses Staphylococcus aureus-induced CD80, CD86, and pro-inflammatory cytokine expression in human B cells

Journal: Arthritis Research & Therapy

doi: 10.1186/s13075-020-2138-x

CTLA-4-Ig binds human memory and activated B cells. a CTLA-4-Ig binds peripheral blood memory B cells. Human peripheral blood mononuclear cells (PBMCs) were stained with 10 μg/ml biotinylated-CTLA-4-Ig or control-protein (Ctrl-Ig), followed by streptavidin-PE together with anti-CD20 and anti-CD27 antibodies and analyzed using flow cytometry. Left histograms, one representative binding of CTLA-4-Ig gated on CD20 + CD27 − naïve B cells (bottom) or on CD20 + CD27 + memory B cells (top) is shown ( n = 3). Gray peaks, Ctrl-Ig; blue line, CTLA-4-Ig. Middle and right histograms, anti-CD80 or anti-CD86 antibody was used to examine CD80 and CD86 expression on naïve and memory B cells. Gray peak, isotype control; blue line, anti-CD80 or anti-CD86 antibody. b CD19 + B cells were purified from the blood of healthy donors and the purity was determined by anti-CD20 antibody staining. On representative result is shown. c CTLA-4-Ig binds activated B cells. Purified CD19 + B cells from blood were stimulated with anti-IgM and anti-CD40 antibodies for 3 days and the levels of CTLA-4-Ig binding (left), CD80 (middle), and CD86 (right) were examined as described in a . Gray peaks, Ctrl-Ig (10 μg/ml) or isotype control; blue line, CTLA-4-Ig (10 μg/ml) or anti-CD80/86 antibody. Data are representative of three independent experiments
Figure Legend Snippet: CTLA-4-Ig binds human memory and activated B cells. a CTLA-4-Ig binds peripheral blood memory B cells. Human peripheral blood mononuclear cells (PBMCs) were stained with 10 μg/ml biotinylated-CTLA-4-Ig or control-protein (Ctrl-Ig), followed by streptavidin-PE together with anti-CD20 and anti-CD27 antibodies and analyzed using flow cytometry. Left histograms, one representative binding of CTLA-4-Ig gated on CD20 + CD27 − naïve B cells (bottom) or on CD20 + CD27 + memory B cells (top) is shown ( n = 3). Gray peaks, Ctrl-Ig; blue line, CTLA-4-Ig. Middle and right histograms, anti-CD80 or anti-CD86 antibody was used to examine CD80 and CD86 expression on naïve and memory B cells. Gray peak, isotype control; blue line, anti-CD80 or anti-CD86 antibody. b CD19 + B cells were purified from the blood of healthy donors and the purity was determined by anti-CD20 antibody staining. On representative result is shown. c CTLA-4-Ig binds activated B cells. Purified CD19 + B cells from blood were stimulated with anti-IgM and anti-CD40 antibodies for 3 days and the levels of CTLA-4-Ig binding (left), CD80 (middle), and CD86 (right) were examined as described in a . Gray peaks, Ctrl-Ig (10 μg/ml) or isotype control; blue line, CTLA-4-Ig (10 μg/ml) or anti-CD80/86 antibody. Data are representative of three independent experiments

Techniques Used: Staining, Flow Cytometry, Binding Assay, Expressing, Purification

15) Product Images from "ECIS technology reveals that monocytes isolated by CD14+ve selection mediate greater loss of BBB integrity than untouched monocytes, which occurs to a greater extent with IL-1β activated endothelium in comparison to TNFα"

Article Title: ECIS technology reveals that monocytes isolated by CD14+ve selection mediate greater loss of BBB integrity than untouched monocytes, which occurs to a greater extent with IL-1β activated endothelium in comparison to TNFα

Journal: PLoS ONE

doi: 10.1371/journal.pone.0180267

Analysis of enriched monocyte purity comparing the two monocyte isolation methods conducted in parallel. (A) Representative forward and side scatter flow diagrams (n = 5) of PBMC (left), CD14 positively isolated monocyte (centre) and untouched monocyte (right) from the same donor. (B) Comparison of monocyte subsets (red) and lymphocyte (blue) in PBMCs and in enriched monocytes, identified using PE-conjugated anti-CD14 antibody and APC-conjugated anti-CD16 antibody. (C) Monocyte and lymphocyte gating strategy highlighting the monocyte singlet gates and doublets to emphasise the difference between the two methods. More extensive gating showing the hierarchical strategy for defining the singlet gates is shown in S4 Fig .
Figure Legend Snippet: Analysis of enriched monocyte purity comparing the two monocyte isolation methods conducted in parallel. (A) Representative forward and side scatter flow diagrams (n = 5) of PBMC (left), CD14 positively isolated monocyte (centre) and untouched monocyte (right) from the same donor. (B) Comparison of monocyte subsets (red) and lymphocyte (blue) in PBMCs and in enriched monocytes, identified using PE-conjugated anti-CD14 antibody and APC-conjugated anti-CD16 antibody. (C) Monocyte and lymphocyte gating strategy highlighting the monocyte singlet gates and doublets to emphasise the difference between the two methods. More extensive gating showing the hierarchical strategy for defining the singlet gates is shown in S4 Fig .

Techniques Used: Isolation, Flow Cytometry

16) Product Images from "Immunomodulation by memantine in therapy of Alzheimer's disease is mediated through inhibition of Kv1.3 channels and T cell responsiveness"

Article Title: Immunomodulation by memantine in therapy of Alzheimer's disease is mediated through inhibition of Kv1.3 channels and T cell responsiveness

Journal: Oncotarget

doi: 10.18632/oncotarget.10777

Memantine abrogates T-bet expression and proliferation of human T cells A. Human peripheral blood CD3 + T cells were stimulated with CD3 Abs (left), PMA/IO (middle) or irradiated, CD3-depleted PBMCs from another healthy donor in MLRs (right) +/- memantine. DNA synthesis was determined by 3 [H]-Thymidine incorporation (cpm). Upper graphs represent a single experiment and lower graphs the mean relative proliferation + SD; n=4-6 and MLR n=3. B. T-bet expression in T cells reacting in MLRs +/- memantine was determined by flow cytometry. The histogram displays a representative experiment and data in the graph the mean + SD percentage of T-bet + cells; n=5. C. The proliferation of naïve CD45RO - and memory CD45RO + CD4 + human T cells was analyzed in MLRs +/- memantine. Graphs show the data of a representative experiment (left) and the mean relative proliferation + SD of 5 experiments (right). The significance of data was determined with Student's t test; p*
Figure Legend Snippet: Memantine abrogates T-bet expression and proliferation of human T cells A. Human peripheral blood CD3 + T cells were stimulated with CD3 Abs (left), PMA/IO (middle) or irradiated, CD3-depleted PBMCs from another healthy donor in MLRs (right) +/- memantine. DNA synthesis was determined by 3 [H]-Thymidine incorporation (cpm). Upper graphs represent a single experiment and lower graphs the mean relative proliferation + SD; n=4-6 and MLR n=3. B. T-bet expression in T cells reacting in MLRs +/- memantine was determined by flow cytometry. The histogram displays a representative experiment and data in the graph the mean + SD percentage of T-bet + cells; n=5. C. The proliferation of naïve CD45RO - and memory CD45RO + CD4 + human T cells was analyzed in MLRs +/- memantine. Graphs show the data of a representative experiment (left) and the mean relative proliferation + SD of 5 experiments (right). The significance of data was determined with Student's t test; p*

Techniques Used: Expressing, Irradiation, DNA Synthesis, Flow Cytometry, Cytometry

17) Product Images from "In vitro and in vivo antivirus activity of an anti-programmed death-ligand 1 (PD-L1) rat-bovine chimeric antibody against bovine leukemia virus infection"

Article Title: In vitro and in vivo antivirus activity of an anti-programmed death-ligand 1 (PD-L1) rat-bovine chimeric antibody against bovine leukemia virus infection

Journal: PLoS ONE

doi: 10.1371/journal.pone.0174916

Activation of T-cell proliferation and interferon-γ (IFN-γ) production by Boch4G12. (A and B) The proliferation of CD4 + and CD8 + cells in purified bovine CD3 + T cells. Peripheral blood mononuclear cells (PBMCs) isolated from healthy cattle were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), stimulated with 1 μg/ml anti-CD3 and anti-CD28 antibodies, and then cultured for 3 days with enhanced green fluorescent protein (EGFP) cells or programmed death-ligand 1 (PD-L1)/EGFP cells without antibodies (A), or PD-L1/EGFP cells in the presence of 10 μg/ml bovine IgG control or Boch4G12 (B). n = 10 and 6, respectively; A P -value less than 0.05 was considered statistically significant. *, P
Figure Legend Snippet: Activation of T-cell proliferation and interferon-γ (IFN-γ) production by Boch4G12. (A and B) The proliferation of CD4 + and CD8 + cells in purified bovine CD3 + T cells. Peripheral blood mononuclear cells (PBMCs) isolated from healthy cattle were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), stimulated with 1 μg/ml anti-CD3 and anti-CD28 antibodies, and then cultured for 3 days with enhanced green fluorescent protein (EGFP) cells or programmed death-ligand 1 (PD-L1)/EGFP cells without antibodies (A), or PD-L1/EGFP cells in the presence of 10 μg/ml bovine IgG control or Boch4G12 (B). n = 10 and 6, respectively; A P -value less than 0.05 was considered statistically significant. *, P

Techniques Used: Activation Assay, Purification, Isolation, Labeling, Cell Culture

18) Product Images from "Long Noncoding RNAs and mRNA Regulation in Peripheral Blood Mononuclear Cells of Patients with Chronic Obstructive Pulmonary Disease"

Article Title: Long Noncoding RNAs and mRNA Regulation in Peripheral Blood Mononuclear Cells of Patients with Chronic Obstructive Pulmonary Disease

Journal: Mediators of Inflammation

doi: 10.1155/2018/7501851

Expression of lncRNAs in the isolated different cell types of PBMCs from smokers (a) and COPD patients (b). The expression of NR_026891.1 , ENST00000502883.1 , HIT000648516 , XR_429541.1 , and ENST00000597550.1 was examined by qRT-PCR on CD4 + T lymphocytes, CD8 + T lymphocytes, CD14 + monocytes, and CD20 + B lymphocytes from smokers and COPD patients. Data are presented as 2 −ΔΔCt relative to β -actin.
Figure Legend Snippet: Expression of lncRNAs in the isolated different cell types of PBMCs from smokers (a) and COPD patients (b). The expression of NR_026891.1 , ENST00000502883.1 , HIT000648516 , XR_429541.1 , and ENST00000597550.1 was examined by qRT-PCR on CD4 + T lymphocytes, CD8 + T lymphocytes, CD14 + monocytes, and CD20 + B lymphocytes from smokers and COPD patients. Data are presented as 2 −ΔΔCt relative to β -actin.

Techniques Used: Expressing, Isolation, Quantitative RT-PCR

Hierarchical clustering, scatter plot result, and Venn diagram of differentially expressed lncRNAs and mRNAs in PBMCs from nonsmokers, smokers, and COPD patients. (a) Hierarchical clustering image of lncRNA expression of pooled RNA samples from PBMCs. (b) Scatter plot of lncRNA expression of PBMCs. (c) Hierarchical clustering image of mRNA expression of pooled RNA samples from PBMCs. (d) Scatter plot of mRNA expression of PBMCs. Red and green colored dots represent up- and downregulated miRNAs in scatter plot, respectively. (e) Venn diagram of differentially expressed lncRNAs and mRNAs. Figure reproduced from Dang et al. [ 10 ], under the Creative Commons Attribution License/public domain.
Figure Legend Snippet: Hierarchical clustering, scatter plot result, and Venn diagram of differentially expressed lncRNAs and mRNAs in PBMCs from nonsmokers, smokers, and COPD patients. (a) Hierarchical clustering image of lncRNA expression of pooled RNA samples from PBMCs. (b) Scatter plot of lncRNA expression of PBMCs. (c) Hierarchical clustering image of mRNA expression of pooled RNA samples from PBMCs. (d) Scatter plot of mRNA expression of PBMCs. Red and green colored dots represent up- and downregulated miRNAs in scatter plot, respectively. (e) Venn diagram of differentially expressed lncRNAs and mRNAs. Figure reproduced from Dang et al. [ 10 ], under the Creative Commons Attribution License/public domain.

Techniques Used: Expressing

19) Product Images from "Empty conformers of HLA-B preferentially bind CD8 and regulate CD8+ T cell function"

Article Title: Empty conformers of HLA-B preferentially bind CD8 and regulate CD8+ T cell function

Journal: eLife

doi: 10.7554/eLife.36341

Clustering of peptide-deficient HLA-I in cognate peptide-induced immunological synapses. CTL line B8-RL8 was incubated with activated PBMCs from Donor 25 (carrying B*08:01 and B*35:01) loaded with peptide RL8 ( A–D ) or not ( E and F ). Cells were fixed and stained with anti-CD8 and W6/32 or HC10 before analysis by confocal microscopy. Peptides were used at a concentration of 100 μM. Anti-CD8 staining ( A , C and E ) is shown in red and W6/32 ( B ) or HC10 ( D and F ) in green. Arrowheads indicate peptide-deficient conformers of HLA-I clustering at the interface between PBMCs and CTL line. The intensity of HLA-I staining of the PBMCs at the interface was compared with the membrane at a noncontact area and plotted as the fold increase above background ( G ). The results with a total of 39 conjugates (for B and D ) or 10 conjugates (for F ) per condition are shown. CD8 clustering was derived from 39 conjugates as condition shown in C. The mean ± SEM was shown. Statistical analyses were undertaken using one-way ANOVA analysis with Fisher’s LSD test. ****, p
Figure Legend Snippet: Clustering of peptide-deficient HLA-I in cognate peptide-induced immunological synapses. CTL line B8-RL8 was incubated with activated PBMCs from Donor 25 (carrying B*08:01 and B*35:01) loaded with peptide RL8 ( A–D ) or not ( E and F ). Cells were fixed and stained with anti-CD8 and W6/32 or HC10 before analysis by confocal microscopy. Peptides were used at a concentration of 100 μM. Anti-CD8 staining ( A , C and E ) is shown in red and W6/32 ( B ) or HC10 ( D and F ) in green. Arrowheads indicate peptide-deficient conformers of HLA-I clustering at the interface between PBMCs and CTL line. The intensity of HLA-I staining of the PBMCs at the interface was compared with the membrane at a noncontact area and plotted as the fold increase above background ( G ). The results with a total of 39 conjugates (for B and D ) or 10 conjugates (for F ) per condition are shown. CD8 clustering was derived from 39 conjugates as condition shown in C. The mean ± SEM was shown. Statistical analyses were undertaken using one-way ANOVA analysis with Fisher’s LSD test. ****, p

Techniques Used: CTL Assay, Incubation, Staining, Confocal Microscopy, Concentration Assay, Derivative Assay

20) Product Images from "Structural Influence on the Dominance of Virus-Specific CD4 T Cell Epitopes in Zika Virus Infection"

Article Title: Structural Influence on the Dominance of Virus-Specific CD4 T Cell Epitopes in Zika Virus Infection

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01196

CD4 T cell responses to Zika virus infection. (A) Schematics of a flavivirus particle representing an immature (left) and mature (right) virion that comprises three structural proteins: C (capsid), prM (membrane), and E (envelope). (B) Representative FACS plot from CD8-depleted peripheral blood mononuclear cells (PBMCs). (C) Individual CD4 T cell responses to C, prM, and E from Zika patients and naïve individuals as determined in IL-2 ELISPOT assays. Results are given as spot-forming cells (SFCs). Medians are depicted as black lines. The dashed line represents the cutoff for assay positivity. (D) Percentage of spots contributed by C, prM, and E peptides in Zika patients.
Figure Legend Snippet: CD4 T cell responses to Zika virus infection. (A) Schematics of a flavivirus particle representing an immature (left) and mature (right) virion that comprises three structural proteins: C (capsid), prM (membrane), and E (envelope). (B) Representative FACS plot from CD8-depleted peripheral blood mononuclear cells (PBMCs). (C) Individual CD4 T cell responses to C, prM, and E from Zika patients and naïve individuals as determined in IL-2 ELISPOT assays. Results are given as spot-forming cells (SFCs). Medians are depicted as black lines. The dashed line represents the cutoff for assay positivity. (D) Percentage of spots contributed by C, prM, and E peptides in Zika patients.

Techniques Used: Infection, FACS, Enzyme-linked Immunospot

21) Product Images from "CXCL12-Induced Monocyte-Endothelial Interactions Promote Lymphocyte Transmigration Across an in Vitro Blood-Brain Barrier"

Article Title: CXCL12-Induced Monocyte-Endothelial Interactions Promote Lymphocyte Transmigration Across an in Vitro Blood-Brain Barrier

Journal: Science translational medicine

doi: 10.1126/scitranslmed.3003197

Apical CXCL12 induced PBMC transmigration. ( A ) PBMCs from human subjects were stained with fluorescence-conjugated antibodies against CD4, CD8, CD14, CD19, and CXCR4 (12G5), and analyzed by flow cytometry. Percentages of CXCR4-positive cells in each subpopulation
Figure Legend Snippet: Apical CXCL12 induced PBMC transmigration. ( A ) PBMCs from human subjects were stained with fluorescence-conjugated antibodies against CD4, CD8, CD14, CD19, and CXCR4 (12G5), and analyzed by flow cytometry. Percentages of CXCR4-positive cells in each subpopulation

Techniques Used: Transmigration Assay, Staining, Fluorescence, Flow Cytometry, Cytometry

CXCL12-induced monocyte-endothelial cell interactions modulate lymphocyte transmigration. ( A ) Lymphocytes were isolated from PBMCs using negative selection. Isolated lymphocytes were stained for CD4, CD8, CD14, and CD19 markers to verify purity. ( B ) Monocytes
Figure Legend Snippet: CXCL12-induced monocyte-endothelial cell interactions modulate lymphocyte transmigration. ( A ) Lymphocytes were isolated from PBMCs using negative selection. Isolated lymphocytes were stained for CD4, CD8, CD14, and CD19 markers to verify purity. ( B ) Monocytes

Techniques Used: Transmigration Assay, Isolation, Selection, Staining

22) Product Images from "Immunosuppressive CD14+HLA-DRlow/− Monocytes in Prostate Cancer"

Article Title: Immunosuppressive CD14+HLA-DRlow/− Monocytes in Prostate Cancer

Journal: The Prostate

doi: 10.1002/pros.21078

T cells in PBMCs of subjects described in the legend to . a: Percent of CD4 + cells (circles) and CD8 + cells (squares) among CD3 + cells. The percentage of CD4 + cells in AMC differed statistically from uPCa( P
Figure Legend Snippet: T cells in PBMCs of subjects described in the legend to . a: Percent of CD4 + cells (circles) and CD8 + cells (squares) among CD3 + cells. The percentage of CD4 + cells in AMC differed statistically from uPCa( P

Techniques Used:

23) Product Images from "Modulation of Type I Interferon-Associated Viral Sensing during Acute Simian Immunodeficiency Virus Infection in African Green Monkeys"

Article Title: Modulation of Type I Interferon-Associated Viral Sensing during Acute Simian Immunodeficiency Virus Infection in African Green Monkeys

Journal: Journal of Virology

doi: 10.1128/JVI.02430-14

(A) Identification of cell types producing IFN-I upon HSV stimulation. (A) BDCA-2 expression on IFN-α2 + cells. PBMCs from a representative animal stimulated with HSV at day 2 p.i. are shown. From the CD123 + IFN-α + (gray) and the CD123
Figure Legend Snippet: (A) Identification of cell types producing IFN-I upon HSV stimulation. (A) BDCA-2 expression on IFN-α2 + cells. PBMCs from a representative animal stimulated with HSV at day 2 p.i. are shown. From the CD123 + IFN-α + (gray) and the CD123

Techniques Used: Expressing

24) Product Images from "Characterization of CD4 T Cell Epitopes of Infliximab and Rituximab Identified from Healthy Donors"

Article Title: Characterization of CD4 T Cell Epitopes of Infliximab and Rituximab Identified from Healthy Donors

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00500

Specificity and restriction of T cell lines generated from healthy donors . (A) CD4 T cell lines were generated in vitro by four weekly rounds of stimulation with autologous dendritic cells loaded with either rituximab (Rtx) or infliximab (Ifx). T cells were incubated with autologous unloaded peripheral blood mononuclear cells (PBMCs) (none) and with PBMCs loaded with individual peptides. Activation of the T cells was evaluated by interferon-γ (IFN-γ) ELISPOT, and peptide specificity was confirmed in two independent experiments. Each panel reports the peptide-specific T cell lines found from one donor. Cells from donors 90, 97, 136, and 201 were used to evaluate the T cell response to Rtx, while cells from donors 221, 241, 244, and 251 were used for Ifx. (B) For inhibition assays, anti-HLA-DR (L243), -DQ (SPVL3), and -DP (B7/21) antibodies were added at 10 µg/mL to the IFN-γ ELISPOT.
Figure Legend Snippet: Specificity and restriction of T cell lines generated from healthy donors . (A) CD4 T cell lines were generated in vitro by four weekly rounds of stimulation with autologous dendritic cells loaded with either rituximab (Rtx) or infliximab (Ifx). T cells were incubated with autologous unloaded peripheral blood mononuclear cells (PBMCs) (none) and with PBMCs loaded with individual peptides. Activation of the T cells was evaluated by interferon-γ (IFN-γ) ELISPOT, and peptide specificity was confirmed in two independent experiments. Each panel reports the peptide-specific T cell lines found from one donor. Cells from donors 90, 97, 136, and 201 were used to evaluate the T cell response to Rtx, while cells from donors 221, 241, 244, and 251 were used for Ifx. (B) For inhibition assays, anti-HLA-DR (L243), -DQ (SPVL3), and -DP (B7/21) antibodies were added at 10 µg/mL to the IFN-γ ELISPOT.

Techniques Used: Generated, In Vitro, Incubation, Activation Assay, Enzyme-linked Immunospot, Inhibition

Identification of CD4 T cell epitopes of infliximab (Ifx) and rituximab (Rtx) from healthy donors . CD4 T cell lines were generated in vitro by four weekly rounds of stimulation with autologous dendritic cells (DCs) loaded with either Rtx or Ifx. Specificity of CD4 T cell lines #113.6 raised against Rtx (A) and #247.26 raised against Ifx (B) was analyzed by interferon-γ ELISPOT. T cells were incubated with autologous DCs alone (none) or with DCs previously loaded with each antibody (3 µM) or with autologous unloaded peripheral blood mononuclear cells (PBMCs) (no pool) or PBMCs loaded with a pool of peptides (10 µg/mL) (left panels) and with individual peptides (right panels).
Figure Legend Snippet: Identification of CD4 T cell epitopes of infliximab (Ifx) and rituximab (Rtx) from healthy donors . CD4 T cell lines were generated in vitro by four weekly rounds of stimulation with autologous dendritic cells (DCs) loaded with either Rtx or Ifx. Specificity of CD4 T cell lines #113.6 raised against Rtx (A) and #247.26 raised against Ifx (B) was analyzed by interferon-γ ELISPOT. T cells were incubated with autologous DCs alone (none) or with DCs previously loaded with each antibody (3 µM) or with autologous unloaded peripheral blood mononuclear cells (PBMCs) (no pool) or PBMCs loaded with a pool of peptides (10 µg/mL) (left panels) and with individual peptides (right panels).

Techniques Used: Generated, In Vitro, Enzyme-linked Immunospot, Incubation

Identification of CD4 T cell epitopes of infliximab (Ifx) and rituximab (Rtx) from healthy donors . CD4 T cell lines were generated in vitro by four weekly rounds of stimulation with autologous dendritic cells (DCs) loaded with either Rtx or Ifx. Specificity of CD4 T cell lines #113.6 raised against Rtx (A) and #247.26 raised against Ifx (B) was analyzed by interferon-γ ELISPOT. T cells were incubated with autologous DCs alone (none) or with DCs previously loaded with each antibody (3 µM) or with autologous unloaded peripheral blood mononuclear cells (PBMCs) (no pool) or PBMCs loaded with a pool of peptides (10 µg/mL) (left panels) and with individual peptides (right panels).
Figure Legend Snippet: Identification of CD4 T cell epitopes of infliximab (Ifx) and rituximab (Rtx) from healthy donors . CD4 T cell lines were generated in vitro by four weekly rounds of stimulation with autologous dendritic cells (DCs) loaded with either Rtx or Ifx. Specificity of CD4 T cell lines #113.6 raised against Rtx (A) and #247.26 raised against Ifx (B) was analyzed by interferon-γ ELISPOT. T cells were incubated with autologous DCs alone (none) or with DCs previously loaded with each antibody (3 µM) or with autologous unloaded peripheral blood mononuclear cells (PBMCs) (no pool) or PBMCs loaded with a pool of peptides (10 µg/mL) (left panels) and with individual peptides (right panels).

Techniques Used: Generated, In Vitro, Enzyme-linked Immunospot, Incubation

25) Product Images from "CD1a presentation of endogenous antigens by group 2 innate lymphoid cells"

Article Title: CD1a presentation of endogenous antigens by group 2 innate lymphoid cells

Journal: Science immunology

doi: 10.1126/sciimmunol.aan5918

ILC2 present bacterial lipid ligands derived from Staphylococcus aureus A. and B. Autologous ILC2 and T cells were isolated from donor PBMCs by flow cytometric sorting and CD3 MACS bead separation respectively. Prior to co-culture with autologous T cells, ILC2 were pulsed with heat-killed S. aureus preparation (HKSA). IFNγ (A.) (p
Figure Legend Snippet: ILC2 present bacterial lipid ligands derived from Staphylococcus aureus A. and B. Autologous ILC2 and T cells were isolated from donor PBMCs by flow cytometric sorting and CD3 MACS bead separation respectively. Prior to co-culture with autologous T cells, ILC2 were pulsed with heat-killed S. aureus preparation (HKSA). IFNγ (A.) (p

Techniques Used: Derivative Assay, Isolation, Flow Cytometry, Magnetic Cell Separation, Co-Culture Assay

ILC2 present HDM-derived lipid ligands to CD1a responsive T cells. Autologous ILC2 and T cells were isolated from donor PBMCs by fluorescence activated cell sorting and CD3 MACS microbead separation respectively. A. and B. Prior to co-culture with autologous T cells, ILC2 were pulsed with HDM extract (7 µg / ml), and IFNγ (A.) and IL-22 (B.) production was detected by ELISpot in the absence or presence of 10 µg / ml anti-CD1a blocking antibody or isotype control. In addition ILC2 alone were stimulated with PMA (15 ng / ml) and Ionomycin (7.5 ng / ml) (P/I). ( A. p
Figure Legend Snippet: ILC2 present HDM-derived lipid ligands to CD1a responsive T cells. Autologous ILC2 and T cells were isolated from donor PBMCs by fluorescence activated cell sorting and CD3 MACS microbead separation respectively. A. and B. Prior to co-culture with autologous T cells, ILC2 were pulsed with HDM extract (7 µg / ml), and IFNγ (A.) and IL-22 (B.) production was detected by ELISpot in the absence or presence of 10 µg / ml anti-CD1a blocking antibody or isotype control. In addition ILC2 alone were stimulated with PMA (15 ng / ml) and Ionomycin (7.5 ng / ml) (P/I). ( A. p

Techniques Used: Derivative Assay, Isolation, Fluorescence, FACS, Magnetic Cell Separation, Co-Culture Assay, Enzyme-linked Immunospot, Blocking Assay

ILC2 express PLA2G4A which generates CD1a ligands. A. PLA2 gene expression analysis of skin and blood derived ILC2 and T cells determined by RNA Sequencing and measured in Reads Per Kilobase of transcript per Million mapped reads (RPKM). B. Cytosolic PLA2 activity of recombinant PLA2G4A irreversibly inhibited by 1 µM MAFP measured using a biochemical assay kit. C. and D. Autologous ILC2 and T cells were isolated from donor PBMCs by fluorescence activated cell sorting and CD3 MACS bead separation respectively. Prior to co-culture with autologous T cells, ILC2 were either unpulsed (U) or pulsed with 1 µg / ml PLA2G4A or PLA2G4A inhibited with 1 µM MAFP. IFNγ (C .) (p
Figure Legend Snippet: ILC2 express PLA2G4A which generates CD1a ligands. A. PLA2 gene expression analysis of skin and blood derived ILC2 and T cells determined by RNA Sequencing and measured in Reads Per Kilobase of transcript per Million mapped reads (RPKM). B. Cytosolic PLA2 activity of recombinant PLA2G4A irreversibly inhibited by 1 µM MAFP measured using a biochemical assay kit. C. and D. Autologous ILC2 and T cells were isolated from donor PBMCs by fluorescence activated cell sorting and CD3 MACS bead separation respectively. Prior to co-culture with autologous T cells, ILC2 were either unpulsed (U) or pulsed with 1 µg / ml PLA2G4A or PLA2G4A inhibited with 1 µM MAFP. IFNγ (C .) (p

Techniques Used: Expressing, Derivative Assay, RNA Sequencing Assay, Activity Assay, Recombinant, Isolation, Fluorescence, FACS, Magnetic Cell Separation, Co-Culture Assay

26) Product Images from "Enhanced killing of chordoma cells by antibody-dependent cell-mediated cytotoxicity employing the novel anti-PD-L1 antibody avelumab"

Article Title: Enhanced killing of chordoma cells by antibody-dependent cell-mediated cytotoxicity employing the novel anti-PD-L1 antibody avelumab

Journal: Oncotarget

doi: 10.18632/oncotarget.9256

Brachyury-specific CD8 + T cells increased chordoma cells' sensitivity to avelumab-mediated ADCC A. Model of indirect enhancement of ADCC by tumor antigen-specific T cells: 1) brachyury-specific T-cell recognition of chordoma tumor; 2) induced IFN-γ secretion; 3) PD-L1 upregulation; 4) increased binding of anti-PD-L1 (avelumab); and 5) enhanced NK cell-mediated killing of tumor (ADCC). UM-Chor1 cells were co-cultured for 24 h with brachyury-specific CD8 + T cells or naïve T cells isolated from PBMCs of a normal donor. As a positive control, UM-Chor1 cells were treated or untreated with 50 ng/mL of IFN-γ for 24 h. B. Concentration of IFN-γ (pg/mL) in supernatant fluid following T cell/tumor cell co-incubation. C. Expression of PD-L1 in UM-Chor1 cells was analyzed by flow cytometry. Values in bold denote an increase of > 10% relative to vehicle control cells. D. ADCC assays were performed using UM-Chor1 cells, with purified normal donor NK cells as effector cells. Select groups of cells were incubated with avelumab. Statistical analyses were done by Student's t test. * = P
Figure Legend Snippet: Brachyury-specific CD8 + T cells increased chordoma cells' sensitivity to avelumab-mediated ADCC A. Model of indirect enhancement of ADCC by tumor antigen-specific T cells: 1) brachyury-specific T-cell recognition of chordoma tumor; 2) induced IFN-γ secretion; 3) PD-L1 upregulation; 4) increased binding of anti-PD-L1 (avelumab); and 5) enhanced NK cell-mediated killing of tumor (ADCC). UM-Chor1 cells were co-cultured for 24 h with brachyury-specific CD8 + T cells or naïve T cells isolated from PBMCs of a normal donor. As a positive control, UM-Chor1 cells were treated or untreated with 50 ng/mL of IFN-γ for 24 h. B. Concentration of IFN-γ (pg/mL) in supernatant fluid following T cell/tumor cell co-incubation. C. Expression of PD-L1 in UM-Chor1 cells was analyzed by flow cytometry. Values in bold denote an increase of > 10% relative to vehicle control cells. D. ADCC assays were performed using UM-Chor1 cells, with purified normal donor NK cells as effector cells. Select groups of cells were incubated with avelumab. Statistical analyses were done by Student's t test. * = P

Techniques Used: Binding Assay, Cell Culture, Isolation, Positive Control, Concentration Assay, Incubation, Expressing, Flow Cytometry, Cytometry, Purification

27) Product Images from "Platelet-Derived Growth Factor-BB Protects Mesenchymal Stem Cells (MSCs) Derived From Immune Thrombocytopenia Patients Against Apoptosis and Senescence and Maintains MSC-Mediated Immunosuppression"

Article Title: Platelet-Derived Growth Factor-BB Protects Mesenchymal Stem Cells (MSCs) Derived From Immune Thrombocytopenia Patients Against Apoptosis and Senescence and Maintains MSC-Mediated Immunosuppression

Journal: Stem Cells Translational Medicine

doi: 10.5966/sctm.2015-0360

MSCs from ITP patients showed impaired immunosuppressive properties. (A): Effects of MSCs on PHA-induced T-cell proliferation. Data expressed as the absorbance at 450 mm ( n = 10). (B): The proportion of CD4+CD25+Foxp3+ Tregs in CD4+ T cells cocultured with MSCs or CD4+ T cells cultured alone detected by flow cytometry ( n = 10). (C): Determination of IFN-γ in coculture system by enzyme-linked immunosorbent assay (ELISA; n = 10). (D): Determination of IL-4 in coculture system by ELISA ( n = 10). (E): Determination of IL-10 in coculture system by ELISA ( n = 10). (F): Effect of MSCs on the antigen (digested GPIIb-IIIa)-induced IgG anti-GPIIb-IIIa antibody production by PBMCs. IgG anti-GPIIb-IIIa antibody levels in undiluted culture supernatants were measured by anti-GPIIb-IIIa ELISA, and the data are presented as absorbance ( n = 10). The MSCs used in each assay were at passage three. *, p
Figure Legend Snippet: MSCs from ITP patients showed impaired immunosuppressive properties. (A): Effects of MSCs on PHA-induced T-cell proliferation. Data expressed as the absorbance at 450 mm ( n = 10). (B): The proportion of CD4+CD25+Foxp3+ Tregs in CD4+ T cells cocultured with MSCs or CD4+ T cells cultured alone detected by flow cytometry ( n = 10). (C): Determination of IFN-γ in coculture system by enzyme-linked immunosorbent assay (ELISA; n = 10). (D): Determination of IL-4 in coculture system by ELISA ( n = 10). (E): Determination of IL-10 in coculture system by ELISA ( n = 10). (F): Effect of MSCs on the antigen (digested GPIIb-IIIa)-induced IgG anti-GPIIb-IIIa antibody production by PBMCs. IgG anti-GPIIb-IIIa antibody levels in undiluted culture supernatants were measured by anti-GPIIb-IIIa ELISA, and the data are presented as absorbance ( n = 10). The MSCs used in each assay were at passage three. *, p

Techniques Used: Cell Culture, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

28) Product Images from "Plasma L-Cystine/L-Glutamate Imbalance Increases Tumor Necrosis Factor-Alpha from CD14+ Circulating Monocytes in Patients with Advanced Cirrhosis"

Article Title: Plasma L-Cystine/L-Glutamate Imbalance Increases Tumor Necrosis Factor-Alpha from CD14+ Circulating Monocytes in Patients with Advanced Cirrhosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0023402

L-Cystine dose-dependently increased TNF-alpha from CD14+ monocytes with LPS under the amino acid environment of patients with advanced cirrhosis. A, Isolated CD14+ monocytes (purity > 90%) were cultured at a density of 2.5×10 5 cells/well in 96-well plates containing in ACM and ACM plus L-Cys (L-Cys : 150 nmol/mL) with 1,000 U/mL M-CSF. One half the amount of culture fluid was exchanged every one day. Cells were maintained for 20 days and the proliferation rate of the cells was measured using CFSE staining. B, Influence of L-Cys on microscopic appearance of monocyte proliferation under serum-free conditions. Day 20, cells in firmly adherent clusters in both ACM and ACM+Cys. C, Monocytes were cultured under CCM, HCM, ACM and ACM plus L-Cys (100–300 nmol/mL). Cells were pre-incubated at a density of 2.5×10 5 cells/well in 96-well flat-bottom plates for 2 hours in each of the media, and 100 ng/mL LPS was added. The supernatants were collected after 24 hours and immediately TNF-alpha was determined by specific cytokine ELISA kits. D,E, Similarly as in Fig. 2C, IL-10, IFN gamma from monocytes and GM-CSF from PBMCs under CCM, HCM, ACM, ACM+Cys (L-Cys 150 nmol/mL) were measured with ELISA. F, Cells were harvested after 24 hours, stained with different mAbs, and analyzed using flow cytometry. Cells were stained with FITC-labeled anti-CD14, -CD80, -CD86, and -HLA-DR. A, B and F results are representative of four experiments from three different donors. C, D and E, Mean ± SEM values from five different donors are shown. C,D,E *, p
Figure Legend Snippet: L-Cystine dose-dependently increased TNF-alpha from CD14+ monocytes with LPS under the amino acid environment of patients with advanced cirrhosis. A, Isolated CD14+ monocytes (purity > 90%) were cultured at a density of 2.5×10 5 cells/well in 96-well plates containing in ACM and ACM plus L-Cys (L-Cys : 150 nmol/mL) with 1,000 U/mL M-CSF. One half the amount of culture fluid was exchanged every one day. Cells were maintained for 20 days and the proliferation rate of the cells was measured using CFSE staining. B, Influence of L-Cys on microscopic appearance of monocyte proliferation under serum-free conditions. Day 20, cells in firmly adherent clusters in both ACM and ACM+Cys. C, Monocytes were cultured under CCM, HCM, ACM and ACM plus L-Cys (100–300 nmol/mL). Cells were pre-incubated at a density of 2.5×10 5 cells/well in 96-well flat-bottom plates for 2 hours in each of the media, and 100 ng/mL LPS was added. The supernatants were collected after 24 hours and immediately TNF-alpha was determined by specific cytokine ELISA kits. D,E, Similarly as in Fig. 2C, IL-10, IFN gamma from monocytes and GM-CSF from PBMCs under CCM, HCM, ACM, ACM+Cys (L-Cys 150 nmol/mL) were measured with ELISA. F, Cells were harvested after 24 hours, stained with different mAbs, and analyzed using flow cytometry. Cells were stained with FITC-labeled anti-CD14, -CD80, -CD86, and -HLA-DR. A, B and F results are representative of four experiments from three different donors. C, D and E, Mean ± SEM values from five different donors are shown. C,D,E *, p

Techniques Used: Isolation, Cell Culture, Staining, Incubation, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Labeling

29) Product Images from "Hormonal Contraception and HIV-1 Infection: Medroxyprogesterone Acetate Suppresses Innate and Adaptive Immune Mechanisms"

Article Title: Hormonal Contraception and HIV-1 Infection: Medroxyprogesterone Acetate Suppresses Innate and Adaptive Immune Mechanisms

Journal: Endocrinology

doi: 10.1210/en.2012-1850

Heat map representation of the effect of MPA and sex steroid hormones on the production of cytokines and chemokines by PBMCs activated with a T cell-activating stimulus. PBMCs or VMMCs were activated with anti–CD2/CD3/CD28-coated microbeads, and
Figure Legend Snippet: Heat map representation of the effect of MPA and sex steroid hormones on the production of cytokines and chemokines by PBMCs activated with a T cell-activating stimulus. PBMCs or VMMCs were activated with anti–CD2/CD3/CD28-coated microbeads, and

Techniques Used:

The effect of estrogen (E2), P4, and MPA on cytokine production by activated mononuclear cells. A–D, PBMCs (A), VMMCs (B), purified CD3 + T cells (C), or CD14 + monocytes (D) obtained from healthy female volunteers were incubated for 24 hours in
Figure Legend Snippet: The effect of estrogen (E2), P4, and MPA on cytokine production by activated mononuclear cells. A–D, PBMCs (A), VMMCs (B), purified CD3 + T cells (C), or CD14 + monocytes (D) obtained from healthy female volunteers were incubated for 24 hours in

Techniques Used: Purification, Incubation

30) Product Images from "Plasmacytoid dendritic cells initiate a complex chemokine and cytokine network and are a viable drug target in chronic HCV patients"

Article Title: Plasmacytoid dendritic cells initiate a complex chemokine and cytokine network and are a viable drug target in chronic HCV patients

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20070814

pDCs secrete robust amounts of TNFα in an IFNα-independent manner. (A) PBMCs were incubated for 5 h with 50 HAU/ml FLU or 10 μg/ml LPS, and brefeldin was added during the final 2 h 30 min. Unstimulated PBMCs were used to establish the gating of TNFα-positive cells (not depicted). (B) PBMCs were stimulated for 5 h with FLU ± anti-IFNAR2. ICCS and surface staining were again performed to evaluate TNFα production in stimulated pDCs. The histograms show TNFα levels in BDCA-2 + BDCA-4 + cells. (C) 22,000 purified pDCs were exposed to the indicated amount of rIFNα2. After 20-h incubation, supernatants were harvested and TNFα level was evaluated by Luminex. For each dataset, results are representative of two independent experiments. (D) A schematic representation of TNFα production by pDCs that are independent of the IFNα pathway.
Figure Legend Snippet: pDCs secrete robust amounts of TNFα in an IFNα-independent manner. (A) PBMCs were incubated for 5 h with 50 HAU/ml FLU or 10 μg/ml LPS, and brefeldin was added during the final 2 h 30 min. Unstimulated PBMCs were used to establish the gating of TNFα-positive cells (not depicted). (B) PBMCs were stimulated for 5 h with FLU ± anti-IFNAR2. ICCS and surface staining were again performed to evaluate TNFα production in stimulated pDCs. The histograms show TNFα levels in BDCA-2 + BDCA-4 + cells. (C) 22,000 purified pDCs were exposed to the indicated amount of rIFNα2. After 20-h incubation, supernatants were harvested and TNFα level was evaluated by Luminex. For each dataset, results are representative of two independent experiments. (D) A schematic representation of TNFα production by pDCs that are independent of the IFNα pathway.

Techniques Used: Incubation, Staining, Purification, Luminex

CCL2 is induced in PBMCs by pDC-derived IFNα. (A) 10 6 PBMCs and PBMCs depleted of pDCs (PBMCs-pDCs) were stimulated for 20 h using 5 μg/ml CpG-2216. Supernatants were harvested, and CCL2 protein levels were evaluated by Luminex. Each dot represents a unique donor ( n = 6), and the bars indicate the mean value. (B) 10 6 PBMCs were exposed to the indicated amount or rIFNα2. After a 20-h incubation, supernatants were harvested and CCL2 levels were evaluated. Error bars indicate one SD. (C) PBMCs were exposed for 5 h to rIFNα2, and then ICCS was performed. CCL2 expression in a defined cell population was evaluated using lineage markers, including CD3, CD14, CD16, CD19, CD56, CD83, and BDCA-2. (D) A schematic representation illustrating the induction of CCL2 by pDC-derived IFNα. For B and C, data are representative of two independent experiments.
Figure Legend Snippet: CCL2 is induced in PBMCs by pDC-derived IFNα. (A) 10 6 PBMCs and PBMCs depleted of pDCs (PBMCs-pDCs) were stimulated for 20 h using 5 μg/ml CpG-2216. Supernatants were harvested, and CCL2 protein levels were evaluated by Luminex. Each dot represents a unique donor ( n = 6), and the bars indicate the mean value. (B) 10 6 PBMCs were exposed to the indicated amount or rIFNα2. After a 20-h incubation, supernatants were harvested and CCL2 levels were evaluated. Error bars indicate one SD. (C) PBMCs were exposed for 5 h to rIFNα2, and then ICCS was performed. CCL2 expression in a defined cell population was evaluated using lineage markers, including CD3, CD14, CD16, CD19, CD56, CD83, and BDCA-2. (D) A schematic representation illustrating the induction of CCL2 by pDC-derived IFNα. For B and C, data are representative of two independent experiments.

Techniques Used: Derivative Assay, Luminex, Incubation, Expressing

pDC-derived IFNα inhibits IL-8 production in monocytes and cDCs. (A) PBMCs were stimulated for 5 h with FLU ± anti-IFNAR2. ICCS and surface staining were performed to evaluate IL-8 production in stimulated pDCs. The plots show IL-8 levels in BDCA-2 + BDCA-4 + cells. (B) Purified pDCs were stimulated for 20 h with 50 HAU/ml FLU ± anti-IFNAR2, and supernatants were harvested and analyzed by Luminex. Data shown are the mean of results obtained from two donors. (C) PBMCs were stimulated for 5 h with 50 HAU/ml FLU ± anti-IFNAR2. ICCS and surface staining were performed to evaluate IL-8 production in monocytes (gated on CD14 + expression), cDCs (identified as BDCA-1 + CD19 − cells), and B cells (gated on CD19 + expression). For each dataset, results are representative of two independent experiments. (D) 5 × 10 5 PBMCs were incubated for 5 h with 50 μg/ml CpG-2216 ± anti-IFNAR2. Supernatants were harvested, and IL-8 levels were evaluated by Luminex. (E) 10 6 PBMCs were cultured in the presence of 1 ng/ml rTNFα (solid line) or media alone (dashed line) in the presence of the increasing amounts of rIFNα. After 20 h, supernatants were harvested and IL-8 levels were evaluated by Luminex. Data show the average values obtained from experiments performed on two healthy donors. (F) A schematic representation illustrating the opposing effects of pDC-derived TNFα and IFNα on IL-8 production.
Figure Legend Snippet: pDC-derived IFNα inhibits IL-8 production in monocytes and cDCs. (A) PBMCs were stimulated for 5 h with FLU ± anti-IFNAR2. ICCS and surface staining were performed to evaluate IL-8 production in stimulated pDCs. The plots show IL-8 levels in BDCA-2 + BDCA-4 + cells. (B) Purified pDCs were stimulated for 20 h with 50 HAU/ml FLU ± anti-IFNAR2, and supernatants were harvested and analyzed by Luminex. Data shown are the mean of results obtained from two donors. (C) PBMCs were stimulated for 5 h with 50 HAU/ml FLU ± anti-IFNAR2. ICCS and surface staining were performed to evaluate IL-8 production in monocytes (gated on CD14 + expression), cDCs (identified as BDCA-1 + CD19 − cells), and B cells (gated on CD19 + expression). For each dataset, results are representative of two independent experiments. (D) 5 × 10 5 PBMCs were incubated for 5 h with 50 μg/ml CpG-2216 ± anti-IFNAR2. Supernatants were harvested, and IL-8 levels were evaluated by Luminex. (E) 10 6 PBMCs were cultured in the presence of 1 ng/ml rTNFα (solid line) or media alone (dashed line) in the presence of the increasing amounts of rIFNα. After 20 h, supernatants were harvested and IL-8 levels were evaluated by Luminex. Data show the average values obtained from experiments performed on two healthy donors. (F) A schematic representation illustrating the opposing effects of pDC-derived TNFα and IFNα on IL-8 production.

Techniques Used: Derivative Assay, Staining, Purification, Luminex, Expressing, Incubation, Cell Culture

pDCs are not infected and not stimulated by replication-competent HCV. (A) pDCs were isolated from fresh PBMCs, and the phenotype was analyzed on samples from healthy control subjects ( n = 4, white bars), NRs ( n = 5, gray bars), and SVRs ( n = 5, black bars). The bars represent the average mean fluorescence intensity of the FL-2 signal. (B) To directly evaluate infectability, 10 4 Huh7.5 cells or BDCA-4–purified pDCs were cultured in 96-well plates ± Rluc-J6-JF. After 12 h, cells were washed 3 times with PBS to remove free and membrane-bound viruses. Cells were replated in complete media, and this was considered as time = 0 h for the experiment. At 0, 24, 48, and 72 h, cells were monitored for luciferase activity. Recombinant IL-3 (10 ng/ml) was added in pDCs, and survival was confirmed by trypan blue exclusion at 72 h. Data shown are representative of two healthy donors. (C) Intracellular staining of claudin-1 was performed on Huh-7.5 cell line and freshly isolated PBMCs (from two healthy donors). Expression of claudin-1 by pDCs was evaluated by looking at BDCA-2 + /BDCA-4 + cells within PBMCs. (D) 10 6 PBMCs were stimulated with media alone, influenza-A/PR8, or J6-JFH. After 5 h, intracellular cytokine staining was performed to evaluate IFNα production. pDCs were identified as BDCA-2 + BDCA-4 + cells within the PBMCs, and IFNα level on the gated cells is shown. Data are representative of experiments performed on PBMCs isolated from three healthy donors with two different viral preparations. These data were confirmed by IFNα Luminex in two additional donors (not depicted).
Figure Legend Snippet: pDCs are not infected and not stimulated by replication-competent HCV. (A) pDCs were isolated from fresh PBMCs, and the phenotype was analyzed on samples from healthy control subjects ( n = 4, white bars), NRs ( n = 5, gray bars), and SVRs ( n = 5, black bars). The bars represent the average mean fluorescence intensity of the FL-2 signal. (B) To directly evaluate infectability, 10 4 Huh7.5 cells or BDCA-4–purified pDCs were cultured in 96-well plates ± Rluc-J6-JF. After 12 h, cells were washed 3 times with PBS to remove free and membrane-bound viruses. Cells were replated in complete media, and this was considered as time = 0 h for the experiment. At 0, 24, 48, and 72 h, cells were monitored for luciferase activity. Recombinant IL-3 (10 ng/ml) was added in pDCs, and survival was confirmed by trypan blue exclusion at 72 h. Data shown are representative of two healthy donors. (C) Intracellular staining of claudin-1 was performed on Huh-7.5 cell line and freshly isolated PBMCs (from two healthy donors). Expression of claudin-1 by pDCs was evaluated by looking at BDCA-2 + /BDCA-4 + cells within PBMCs. (D) 10 6 PBMCs were stimulated with media alone, influenza-A/PR8, or J6-JFH. After 5 h, intracellular cytokine staining was performed to evaluate IFNα production. pDCs were identified as BDCA-2 + BDCA-4 + cells within the PBMCs, and IFNα level on the gated cells is shown. Data are representative of experiments performed on PBMCs isolated from three healthy donors with two different viral preparations. These data were confirmed by IFNα Luminex in two additional donors (not depicted).

Techniques Used: Infection, Isolation, Fluorescence, Purification, Cell Culture, Luciferase, Activity Assay, Recombinant, Staining, Expressing, Luminex

31) Product Images from "IL-2 Inhibition of Th17 Generation Rather Than Induction of Treg Cells Is Impaired in Primary Sjögren’s Syndrome Patients"

Article Title: IL-2 Inhibition of Th17 Generation Rather Than Induction of Treg Cells Is Impaired in Primary Sjögren’s Syndrome Patients

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01755

Increased Th17 cells but no change of Treg cells in primary Sjögren’s syndrome (pSS) patients. (A,B) The percentages of IL-17 + CD4 + T cells in the PBMCs from healthy donors or pSS patients were determined by flow cytometry. Representative plot (A) and histogram analysis (B) were shown. (C) The amount of Il17a mRNA in PBMCs was determined by q-PCR. (D,E) The percentages of Foxp3 + CD4 + T cells in the PBMCs were determined by flow cytometry. Representative plot (D) and histogram analysis (E) were shown. (F) The amount of Foxp3 mRNA in PBMCs from healthy donors or pSS patients was determined by q-PCR. (G) IL-17A levels in the minor salivary gland (MSG) from Sicca or pSS patients were determined by immunohistochemistry. (H–I) The amounts of Il17a (H) and Foxp3 mRNA (I) were determined by q-PCR. (J) Immunofluorescence staining of CD4 and IL-17A in the MSG specimens from pSS patients. Representative images are shown (400×). ** p
Figure Legend Snippet: Increased Th17 cells but no change of Treg cells in primary Sjögren’s syndrome (pSS) patients. (A,B) The percentages of IL-17 + CD4 + T cells in the PBMCs from healthy donors or pSS patients were determined by flow cytometry. Representative plot (A) and histogram analysis (B) were shown. (C) The amount of Il17a mRNA in PBMCs was determined by q-PCR. (D,E) The percentages of Foxp3 + CD4 + T cells in the PBMCs were determined by flow cytometry. Representative plot (D) and histogram analysis (E) were shown. (F) The amount of Foxp3 mRNA in PBMCs from healthy donors or pSS patients was determined by q-PCR. (G) IL-17A levels in the minor salivary gland (MSG) from Sicca or pSS patients were determined by immunohistochemistry. (H–I) The amounts of Il17a (H) and Foxp3 mRNA (I) were determined by q-PCR. (J) Immunofluorescence staining of CD4 and IL-17A in the MSG specimens from pSS patients. Representative images are shown (400×). ** p

Techniques Used: Flow Cytometry, Cytometry, Polymerase Chain Reaction, Immunohistochemistry, Immunofluorescence, Staining

IL-2-induced STAT5 binds to STAT3-binding site in human Il17a locus. (A) H E and IHC staining with anti-p-STAT3 and anti-p-STAT5 antibody on the minor salivary gland (MSG) tissues from 10 primary Sjögren’s syndrome (pSS) patients and 7 Sicca patients. Representative images are shown. (B) The levels of p-STAT3 and p-STAT5 in CD4 + T cells in the PBMCs from healthy donors ( n = 5) or pSS ( n = 5) patients were determined by flow cytometry. Representative plot (left) and histogram analysis (right) were shown. (C–E) Naïve CD4 + T cells were stimulated under Th17 conditions with or without IL-2, then crosslinked, and immunoprecipitated with anti-STAT5 (C) , anti-STAT3 (D) , and anti-H3K4me3 (E) . Immunoprecipitated DNA was amplified by q-PCR and expressed as a percentage to input DNA. (F,G) Human naïve CD4 + T cells were differentiated under Th17 conditions with IL-2 by adding STAT5 inhibitor STAT5-IN-1 (5 µM) for 8 days. The percentages of CD4 + IL-17A + cells were determined by intracellular staining (F) . Histogram analysis of the percentages of the differentiated Th17 cells treated with or without STAT5 inhibitor (G) . P value was determined with unpaired t -test. Data are pooled from two independent experiments (error bars denote SEM). * p
Figure Legend Snippet: IL-2-induced STAT5 binds to STAT3-binding site in human Il17a locus. (A) H E and IHC staining with anti-p-STAT3 and anti-p-STAT5 antibody on the minor salivary gland (MSG) tissues from 10 primary Sjögren’s syndrome (pSS) patients and 7 Sicca patients. Representative images are shown. (B) The levels of p-STAT3 and p-STAT5 in CD4 + T cells in the PBMCs from healthy donors ( n = 5) or pSS ( n = 5) patients were determined by flow cytometry. Representative plot (left) and histogram analysis (right) were shown. (C–E) Naïve CD4 + T cells were stimulated under Th17 conditions with or without IL-2, then crosslinked, and immunoprecipitated with anti-STAT5 (C) , anti-STAT3 (D) , and anti-H3K4me3 (E) . Immunoprecipitated DNA was amplified by q-PCR and expressed as a percentage to input DNA. (F,G) Human naïve CD4 + T cells were differentiated under Th17 conditions with IL-2 by adding STAT5 inhibitor STAT5-IN-1 (5 µM) for 8 days. The percentages of CD4 + IL-17A + cells were determined by intracellular staining (F) . Histogram analysis of the percentages of the differentiated Th17 cells treated with or without STAT5 inhibitor (G) . P value was determined with unpaired t -test. Data are pooled from two independent experiments (error bars denote SEM). * p

Techniques Used: Binding Assay, Immunohistochemistry, Staining, Flow Cytometry, Cytometry, Immunoprecipitation, Amplification, Polymerase Chain Reaction

32) Product Images from "Human CD25+CD4+ T Suppressor Cell Clones Produce Transforming Growth Factor ?, but not Interleukin 10, and Are Distinct from Type 1 T Regulatory Cells"

Article Title: Human CD25+CD4+ T Suppressor Cell Clones Produce Transforming Growth Factor ?, but not Interleukin 10, and Are Distinct from Type 1 T Regulatory Cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20021139

Human CD25 + CD4 + T cells or T cell clones do not express membrane-bound TGF-β. Total PBMCs were stained with anti-CD4, -CD25, and -TGF-β Abs. CD25 + CD4 + or CD25 − CD4 + T cells were gated, and expression of TGF-β (thick line) on the two subsets was compared with staining by the secondary reagent alone (thin line; A, left panel). In parallel, CD25 + CD4 + and CD25 − CD4 + T cells were purified by FACS ® sorting or via positive-selection with microbeads. Purified populations were subsequently stained with anti–TGF-β Abs (thick line) or with the secondary reagent alone (thin line). Results are representative of nine independent experiments. For B, several CD25 + CD4 + T cell clones were tested for expression of membrane-bound TGF-β. Clone 87 is representative of nonanergic clones, clone 3 of anergic, nonsuppressive clones, and clone 42 of anergic and suppressive clones. Results are representative of two independent tests.
Figure Legend Snippet: Human CD25 + CD4 + T cells or T cell clones do not express membrane-bound TGF-β. Total PBMCs were stained with anti-CD4, -CD25, and -TGF-β Abs. CD25 + CD4 + or CD25 − CD4 + T cells were gated, and expression of TGF-β (thick line) on the two subsets was compared with staining by the secondary reagent alone (thin line; A, left panel). In parallel, CD25 + CD4 + and CD25 − CD4 + T cells were purified by FACS ® sorting or via positive-selection with microbeads. Purified populations were subsequently stained with anti–TGF-β Abs (thick line) or with the secondary reagent alone (thin line). Results are representative of nine independent experiments. For B, several CD25 + CD4 + T cell clones were tested for expression of membrane-bound TGF-β. Clone 87 is representative of nonanergic clones, clone 3 of anergic, nonsuppressive clones, and clone 42 of anergic and suppressive clones. Results are representative of two independent tests.

Techniques Used: Clone Assay, Staining, Expressing, Purification, FACS, Selection

Isolation and characterization of human CD25 + CD4 + T cells at the clonal level. CD4 + T cells were isolated from PBMCs, stained with anti-CD4 and anti-CD25 mAbs, and separated into CD25 + CD4 + and CD25 − CD4 + T cells by FACS ® sorting to a purity greater than 98 and 99%, respectively (A). Resting T cell clones were stained with anti-CD4 and -CD25 mAbs 12–14 d after the last restimulation. Numbers on the top left indicate clone number, and on the top right the MFI and percent positive cells (B). Resting T cell clones were also tested for their ability to proliferate in response to anti-CD3 mAbs (10 μg/ml) in the absence or presence of IL-2 (100 U/ml; C), or in response to IL-2, IL-15 (10 ng/ml), or IL-2 and IL-15 together. (D) After 48 h of culture, [ 3 H]thymidine was added for an additional 16 h. For B–D results are representative of at least five independent tests.
Figure Legend Snippet: Isolation and characterization of human CD25 + CD4 + T cells at the clonal level. CD4 + T cells were isolated from PBMCs, stained with anti-CD4 and anti-CD25 mAbs, and separated into CD25 + CD4 + and CD25 − CD4 + T cells by FACS ® sorting to a purity greater than 98 and 99%, respectively (A). Resting T cell clones were stained with anti-CD4 and -CD25 mAbs 12–14 d after the last restimulation. Numbers on the top left indicate clone number, and on the top right the MFI and percent positive cells (B). Resting T cell clones were also tested for their ability to proliferate in response to anti-CD3 mAbs (10 μg/ml) in the absence or presence of IL-2 (100 U/ml; C), or in response to IL-2, IL-15 (10 ng/ml), or IL-2 and IL-15 together. (D) After 48 h of culture, [ 3 H]thymidine was added for an additional 16 h. For B–D results are representative of at least five independent tests.

Techniques Used: Isolation, Staining, FACS, Clone Assay

IL-10–induced anergy does not depend on the presence of CD25 + CD4 + T cells. Total PBMCs or PBMCs depleted of CD25 + cells were stimulated with irradiated CD3-depleted allogeneic APCs for 10 d in the absence or presence of IL-10 (100 U/ml). After 10 d, cells were harvested and left unstimulated (cells) or restimulated with the same allogeneic APCs in the absence of IL-10 (cells + stim). After 48 h of culture, [ 3 H]thymidine was added for an additional 16 h. One representative secondary MLR out of four performed shown in panel A. In B, the average percent inhibition of proliferation induced by IL-10 in primary (I°) and secondary (II°) MLRs performed with total or CD25 − PBMCs as responder cells from four independent experiments is shown.
Figure Legend Snippet: IL-10–induced anergy does not depend on the presence of CD25 + CD4 + T cells. Total PBMCs or PBMCs depleted of CD25 + cells were stimulated with irradiated CD3-depleted allogeneic APCs for 10 d in the absence or presence of IL-10 (100 U/ml). After 10 d, cells were harvested and left unstimulated (cells) or restimulated with the same allogeneic APCs in the absence of IL-10 (cells + stim). After 48 h of culture, [ 3 H]thymidine was added for an additional 16 h. One representative secondary MLR out of four performed shown in panel A. In B, the average percent inhibition of proliferation induced by IL-10 in primary (I°) and secondary (II°) MLRs performed with total or CD25 − PBMCs as responder cells from four independent experiments is shown.

Techniques Used: Irradiation, Inhibition

33) Product Images from "CXCL10 and CCL2 mRNA expression in monocytes is inversely correlated with the HLA-DR lower fraction of monocytes in patients with renal cell carcinoma"

Article Title: CXCL10 and CCL2 mRNA expression in monocytes is inversely correlated with the HLA-DR lower fraction of monocytes in patients with renal cell carcinoma

Journal: Oncology Letters

doi: 10.3892/ol.2016.4132

Percentage of CD14 + HLA-DR low/− monocytes in circulating blood is increased in patients with RCC. (A) Representative FACS analysis of CD14 + HLA-DR low/− monocytes in the peripheral blood mononuclear cells of a healthy control donor and a
Figure Legend Snippet: Percentage of CD14 + HLA-DR low/− monocytes in circulating blood is increased in patients with RCC. (A) Representative FACS analysis of CD14 + HLA-DR low/− monocytes in the peripheral blood mononuclear cells of a healthy control donor and a

Techniques Used: FACS

34) Product Images from "The Syk-Coupled C-Type Lectin Receptors Dectin-2 and Dectin-3 Are Involved in Paracoccidioides brasiliensis Recognition by Human Plasmacytoid Dendritic Cells"

Article Title: The Syk-Coupled C-Type Lectin Receptors Dectin-2 and Dectin-3 Are Involved in Paracoccidioides brasiliensis Recognition by Human Plasmacytoid Dendritic Cells

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00464

Paracoccidioides brasiliensis stimulation induces a mature phenotype in plasmacytoid dendritic cells (pDCs) that become able to promote the activation of CD4 + and CD8 + T lymphocytes. Peripheral blood mononuclear cells (PBMCs) were separated into pDC-positive (pDCs + ) and myeloid dendritic cell (mDC)-positive (mDC + ) population using magnetic beads conjugated to anti-CD304 and anti-CD1c antibodies, respectively. (A,B) For characterization of pDCs activation, the cells were labeled with appropriate titers of anti-CD123, MHC-class II, and CD86 antibodies and then 50,000 events were acquired in the flow cytometer and gated as indicated in the dot plots (A) . As the pDC + population was stained with an specific pDC marker (anti-CD123), the characterization of cell maturation was performed by obtaining the pDCs from the PBMCs following one round of positive selection. Data are the means ± SE of the frequency of double-positive cells from three donors, tested in triplicate (B). (C,D) For coculture experiments, the pDCs and mDCs (1 × 10 5 /well) were cultured with or without P. brasiliensis yeast cells (1 × 10 4 ) and after 2 h they were then placed in cocultured with lymphocytes (1 × 10 6 ), which were previously obtained from PBMCs of the same donor and separated with anti-CD3 magnetic beads. After 5 days of coculture, the lymphocytes were analyzed by flow cytometry using anti-CD4, CD8, CD25, and anti-CD69 antibodies. The control wells contained only CD3 + cells. The lymphocyte population was gated by FSC/SSC analysis and the gated cells were then analyzed for expression of lymphocyte activation markers (C) . Data are the means ± SE of the frequency of single positive (CD4 + /CD8 + ) double-positive cells (CD4 + CD25 + and CD8 + CD69 + ) from three donors, tested in triplicate in cultures with [ (D) , right panel] or without P. brasiliensis yeast cells [ (D) , left panel]. * p
Figure Legend Snippet: Paracoccidioides brasiliensis stimulation induces a mature phenotype in plasmacytoid dendritic cells (pDCs) that become able to promote the activation of CD4 + and CD8 + T lymphocytes. Peripheral blood mononuclear cells (PBMCs) were separated into pDC-positive (pDCs + ) and myeloid dendritic cell (mDC)-positive (mDC + ) population using magnetic beads conjugated to anti-CD304 and anti-CD1c antibodies, respectively. (A,B) For characterization of pDCs activation, the cells were labeled with appropriate titers of anti-CD123, MHC-class II, and CD86 antibodies and then 50,000 events were acquired in the flow cytometer and gated as indicated in the dot plots (A) . As the pDC + population was stained with an specific pDC marker (anti-CD123), the characterization of cell maturation was performed by obtaining the pDCs from the PBMCs following one round of positive selection. Data are the means ± SE of the frequency of double-positive cells from three donors, tested in triplicate (B). (C,D) For coculture experiments, the pDCs and mDCs (1 × 10 5 /well) were cultured with or without P. brasiliensis yeast cells (1 × 10 4 ) and after 2 h they were then placed in cocultured with lymphocytes (1 × 10 6 ), which were previously obtained from PBMCs of the same donor and separated with anti-CD3 magnetic beads. After 5 days of coculture, the lymphocytes were analyzed by flow cytometry using anti-CD4, CD8, CD25, and anti-CD69 antibodies. The control wells contained only CD3 + cells. The lymphocyte population was gated by FSC/SSC analysis and the gated cells were then analyzed for expression of lymphocyte activation markers (C) . Data are the means ± SE of the frequency of single positive (CD4 + /CD8 + ) double-positive cells (CD4 + CD25 + and CD8 + CD69 + ) from three donors, tested in triplicate in cultures with [ (D) , right panel] or without P. brasiliensis yeast cells [ (D) , left panel]. * p

Techniques Used: Activation Assay, Magnetic Beads, Labeling, Flow Cytometry, Cytometry, Staining, Marker, Selection, Cell Culture, Expressing

35) Product Images from "Increased CD8+ T cell responses to apoptotic T cell-associated antigens in multiple sclerosis"

Article Title: Increased CD8+ T cell responses to apoptotic T cell-associated antigens in multiple sclerosis

Journal: Journal of Neuroinflammation

doi: 10.1186/1742-2094-10-94

Detection of peripheral CD8 + T cells specific to apoptotic epitopes in healthy donors and multiple sclerosis patients directly in vivo . (A) Representative flow cytometry analysis of peripheral blood mononuclear cells, from a multiple sclerosis (MS) patient and a healthy donor (HD), were stained with a phycoerythrin-cyanine (PeCy7)-labeled mAb to CD8, allophycocyanin (APC)-labeled-HLA-A*0201 dextramers expressing the indicated apoptotic peptides, and the dump channel APC-Cy7-labeled reagents so as to exclude CD4 + T cells, monocytes, B cells, natural killer (NK) cells and dead cells from the analysis. Contour plot analyses show the percentage of CD8 + dextramer + cells. The percentage of cells is indicated in the appropriate quadrant. (B) Representative flow cytometry analysis of PBMCs stained with a pool of APC-labeled–HLA-A*0201 dextramer expressing relevant peptides, fluorescein isothiocyanate-labeled mAb to CD8, PeCy7-labeled mAb to PD-1, AlexaFluor700-labeled mAb to CD69, PECF594-labeled mAb to HLA-DR, and dump channel labeled reagents. Dot plot analyses show percentages of CD69 + , HLA-DR + , or PD-1 + cells in gated CD8 + dextramer + cells. The percentage of cells is reported in each quadrant. (C) Percentage of CD8 + dextramer + cells specific to a single epitope in 10 HLA-A2 + HD and 15 HLA-A2 + patients (each symbol represents the percentage of CD8 + dextramer + cells specific to a single epitope in HDs or patients). (D) Sum of the percentages of all CD8 + dextramer + T cells detected in the single patient or HD (each symbol represents the sum of percentages of the five dextramers tested in the single individual). (E) Percentages of CD69 + , HLA-DR + , or PD-1 + cells in CD8 + dextramer + T cells from MS patients. Statistical analysis was performed with the Mann-Whitney test. MYH9, non-muscle myosin heavy chain 9; VIME, vimentin; ACTB, actin cytoplasmic 1.
Figure Legend Snippet: Detection of peripheral CD8 + T cells specific to apoptotic epitopes in healthy donors and multiple sclerosis patients directly in vivo . (A) Representative flow cytometry analysis of peripheral blood mononuclear cells, from a multiple sclerosis (MS) patient and a healthy donor (HD), were stained with a phycoerythrin-cyanine (PeCy7)-labeled mAb to CD8, allophycocyanin (APC)-labeled-HLA-A*0201 dextramers expressing the indicated apoptotic peptides, and the dump channel APC-Cy7-labeled reagents so as to exclude CD4 + T cells, monocytes, B cells, natural killer (NK) cells and dead cells from the analysis. Contour plot analyses show the percentage of CD8 + dextramer + cells. The percentage of cells is indicated in the appropriate quadrant. (B) Representative flow cytometry analysis of PBMCs stained with a pool of APC-labeled–HLA-A*0201 dextramer expressing relevant peptides, fluorescein isothiocyanate-labeled mAb to CD8, PeCy7-labeled mAb to PD-1, AlexaFluor700-labeled mAb to CD69, PECF594-labeled mAb to HLA-DR, and dump channel labeled reagents. Dot plot analyses show percentages of CD69 + , HLA-DR + , or PD-1 + cells in gated CD8 + dextramer + cells. The percentage of cells is reported in each quadrant. (C) Percentage of CD8 + dextramer + cells specific to a single epitope in 10 HLA-A2 + HD and 15 HLA-A2 + patients (each symbol represents the percentage of CD8 + dextramer + cells specific to a single epitope in HDs or patients). (D) Sum of the percentages of all CD8 + dextramer + T cells detected in the single patient or HD (each symbol represents the sum of percentages of the five dextramers tested in the single individual). (E) Percentages of CD69 + , HLA-DR + , or PD-1 + cells in CD8 + dextramer + T cells from MS patients. Statistical analysis was performed with the Mann-Whitney test. MYH9, non-muscle myosin heavy chain 9; VIME, vimentin; ACTB, actin cytoplasmic 1.

Techniques Used: In Vivo, Flow Cytometry, Cytometry, Mass Spectrometry, Staining, Labeling, Expressing, MANN-WHITNEY

Polyfunctional CD8 + T effector memory cells specific to apoptotic epitopes in multiple sclerosis patients. (A) Representative flow cytometry analysis of peripheral blood mononuclear cells from a multiple sclerosis (MS) patient. Cells were incubated with or without the relevant peptides plus anti-CD28 mAb for 18 h at 37°C. Then cells were stained with dextramers complexed to corresponding peptides, phycoerythrin-cyanine-labeled mAb to CD8 and the dump channel reagents and processed for the detection of IL-17 and IFN-γ, by intracellular staining (ICS) assay with the relevant mAbs. Dot plot analyses are gated on CD8 + dextramer + cells and show percentages of cytokine-producing cells. The percentage of cells is reported in each quadrant. (B) Percentages of CD8 + dextramer + cells specific to the corresponding peptides producing IL-17, IFN-γ, or both within 18 h of contact with the relevant peptides in the 12 patients analyzed. (C) Representative flow cytometry analysis of an antigen-specific T cell line obtained upon 15 days stimulation with the non-muscle myosin heavy chain 9 (MYH9) 478-486 epitope and IL-2. Cells were stained with mAb to CD8, the indicated dextramers, and were then stimulated or not with the relevant soluble peptide plus anti-CD28 mAb for detecting IL-17 and IFN-γ production by ICS assay, as described above. Contourplot analyses are gated on CD8 + dextramer + cells and show percentages of cytokine-producing cells. Similar results were obtained in six patients tested with different apoptotic epitopes.
Figure Legend Snippet: Polyfunctional CD8 + T effector memory cells specific to apoptotic epitopes in multiple sclerosis patients. (A) Representative flow cytometry analysis of peripheral blood mononuclear cells from a multiple sclerosis (MS) patient. Cells were incubated with or without the relevant peptides plus anti-CD28 mAb for 18 h at 37°C. Then cells were stained with dextramers complexed to corresponding peptides, phycoerythrin-cyanine-labeled mAb to CD8 and the dump channel reagents and processed for the detection of IL-17 and IFN-γ, by intracellular staining (ICS) assay with the relevant mAbs. Dot plot analyses are gated on CD8 + dextramer + cells and show percentages of cytokine-producing cells. The percentage of cells is reported in each quadrant. (B) Percentages of CD8 + dextramer + cells specific to the corresponding peptides producing IL-17, IFN-γ, or both within 18 h of contact with the relevant peptides in the 12 patients analyzed. (C) Representative flow cytometry analysis of an antigen-specific T cell line obtained upon 15 days stimulation with the non-muscle myosin heavy chain 9 (MYH9) 478-486 epitope and IL-2. Cells were stained with mAb to CD8, the indicated dextramers, and were then stimulated or not with the relevant soluble peptide plus anti-CD28 mAb for detecting IL-17 and IFN-γ production by ICS assay, as described above. Contourplot analyses are gated on CD8 + dextramer + cells and show percentages of cytokine-producing cells. Similar results were obtained in six patients tested with different apoptotic epitopes.

Techniques Used: Flow Cytometry, Cytometry, Mass Spectrometry, Incubation, Staining, Labeling

Cross-presentation of naturally processed apoptotic epitopes to the related CD8 + T EM cells by dendritic cells. (A) One representative of five experiments in which peripheral blood mononuclear cells from one MS patient were double-stained with mAb to CD8 and dextramers complexed with the indicated apoptotic epitope. These cells were then cultured with autologous dendritic cells (DCs) that had been pulsed or not with the relevant soluble peptide, apoptotic cloned T cells (ACs = apoptotic cells), ACs previously treated with a negative caspase control (Ctr = Z-FA-FMK), or ACs previously treated with the caspase 3 inhibitor (C3I = Z-DEVD-FMK). After 18 h, CD8 + dextramer + cells were tested for their capacity to produce the different cytokines indicated by the intracellular staining assay. Dot plots are gated on CD8 + dextramer + cells and show percentages of the different cytokine-producing cells in each quadrant. (B) Cumulative experiments in five independent patients, performed as described in (A) , showing the mean percentages of cells producing IL-17 (solid bars) and IFN-γ (open bars) in CD8 + dextramer + cells in response to the indicated stimuli: autologous DCs alone; DCs pulsed with the vimentin (VIME) 78-87 peptide; DCs pulsed with ACs, which had been previously treated with a negative caspase control (Z-FA-FMK); or DCs pulsed with ACs, which had been previously treated with the C3I (Z-DEVD-FMK).
Figure Legend Snippet: Cross-presentation of naturally processed apoptotic epitopes to the related CD8 + T EM cells by dendritic cells. (A) One representative of five experiments in which peripheral blood mononuclear cells from one MS patient were double-stained with mAb to CD8 and dextramers complexed with the indicated apoptotic epitope. These cells were then cultured with autologous dendritic cells (DCs) that had been pulsed or not with the relevant soluble peptide, apoptotic cloned T cells (ACs = apoptotic cells), ACs previously treated with a negative caspase control (Ctr = Z-FA-FMK), or ACs previously treated with the caspase 3 inhibitor (C3I = Z-DEVD-FMK). After 18 h, CD8 + dextramer + cells were tested for their capacity to produce the different cytokines indicated by the intracellular staining assay. Dot plots are gated on CD8 + dextramer + cells and show percentages of the different cytokine-producing cells in each quadrant. (B) Cumulative experiments in five independent patients, performed as described in (A) , showing the mean percentages of cells producing IL-17 (solid bars) and IFN-γ (open bars) in CD8 + dextramer + cells in response to the indicated stimuli: autologous DCs alone; DCs pulsed with the vimentin (VIME) 78-87 peptide; DCs pulsed with ACs, which had been previously treated with a negative caspase control (Z-FA-FMK); or DCs pulsed with ACs, which had been previously treated with the C3I (Z-DEVD-FMK).

Techniques Used: Mass Spectrometry, Staining, Cell Culture, Clone Assay

36) Product Images from "TLR7 agonists induce transient viremia and reduce the viral reservoir in SIV-infected rhesus macaques on antiretroviral therapy"

Article Title: TLR7 agonists induce transient viremia and reduce the viral reservoir in SIV-infected rhesus macaques on antiretroviral therapy

Journal: Science translational medicine

doi: 10.1126/scitranslmed.aao4521

Number of CD8 + lymphocytes and plasma SIV RNA kinetics after in vivo CD8 + cell depletion ( A ) CD8 + lymphocytes were depleted in vivo by infusion of monoclonal antibody MT807R1, and numbers of CD8 + cells per microliter of whole blood were monitored by flow cytometry for four macaques (two aviremic and two viremic). Log plasma virus RNA was assessed between days 0 and 35 after CD8 + cell depletion. ( B ) Four SIV-naïve rhesus macaques served as recipients for adoptive transfer of cells from two donor rhesus macaques in remission. Left: In the first transfer, about 50 million frozen PBMCs and LNMCs from donor animals 177-10 and 344-10, isolated after the initiation of ART but before TLR7 agonist treatment, were used for adoptive transfer. For each donor rhesus macaque (177-10, black; 344-10, blue), PBMCs and LNMCs were thawed, combined, and then infused into two naïve rhesus macaques. Right: In the second transfer, about 120 million fresh PBMCs and LNMCs from each donor were isolated at 448 days after cessation of ART. PBMCs and LNMCs were combined and then infused into two additional naïve rhesus macaques. Log plasma virus RNA was assessed between days 0 and 28 after adoptive transfer.
Figure Legend Snippet: Number of CD8 + lymphocytes and plasma SIV RNA kinetics after in vivo CD8 + cell depletion ( A ) CD8 + lymphocytes were depleted in vivo by infusion of monoclonal antibody MT807R1, and numbers of CD8 + cells per microliter of whole blood were monitored by flow cytometry for four macaques (two aviremic and two viremic). Log plasma virus RNA was assessed between days 0 and 35 after CD8 + cell depletion. ( B ) Four SIV-naïve rhesus macaques served as recipients for adoptive transfer of cells from two donor rhesus macaques in remission. Left: In the first transfer, about 50 million frozen PBMCs and LNMCs from donor animals 177-10 and 344-10, isolated after the initiation of ART but before TLR7 agonist treatment, were used for adoptive transfer. For each donor rhesus macaque (177-10, black; 344-10, blue), PBMCs and LNMCs were thawed, combined, and then infused into two naïve rhesus macaques. Right: In the second transfer, about 120 million fresh PBMCs and LNMCs from each donor were isolated at 448 days after cessation of ART. PBMCs and LNMCs were combined and then infused into two additional naïve rhesus macaques. Log plasma virus RNA was assessed between days 0 and 28 after adoptive transfer.

Techniques Used: In Vivo, Flow Cytometry, Cytometry, Adoptive Transfer Assay, Isolation

37) Product Images from "Induction of Immunosuppressive CD8+CD25+FOXP3+ Regulatory T Cells by Suboptimal Stimulation with Staphylococcal Enterotoxin C1"

Article Title: Induction of Immunosuppressive CD8+CD25+FOXP3+ Regulatory T Cells by Suboptimal Stimulation with Staphylococcal Enterotoxin C1

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1602109

Expression of cell surface markers and cytokines related to Tregs by CD8 + CD25 + T cells induced by suboptimal stimulation with SEC1. Human PBMCs depleted of CD25 + T cells were stimulated with a suboptimal concentration of SEC1 (1 ng/ml) for 6 d. Expression of surface markers and cytokines related to Tregs was measured by flow cytometry and a Luminex ELISA kit. ( A ) Percentage (mean ± SEM) of CD8 + CD25 + FOXP3 + T cells expressing surface markers related to Tregs combined from nine independent experiments (three donors). Expression of CTLA-4 was measured by intracellular staining. ( B ) Percentage (mean ± SEM) of CD8 + CD25 + T cells expressing cytokines related to Tregs measured by intracellular staining and flow cytometry. Data are combined from nine independent experiments (three donors). ( C ) During suboptimal stimulation with SEC1, the culture supernatant was collected every 2 d and analyzed using a Milliplex Luminex cytokine analysis kit. Data shown are combined results from six independent experiments (two donors).
Figure Legend Snippet: Expression of cell surface markers and cytokines related to Tregs by CD8 + CD25 + T cells induced by suboptimal stimulation with SEC1. Human PBMCs depleted of CD25 + T cells were stimulated with a suboptimal concentration of SEC1 (1 ng/ml) for 6 d. Expression of surface markers and cytokines related to Tregs was measured by flow cytometry and a Luminex ELISA kit. ( A ) Percentage (mean ± SEM) of CD8 + CD25 + FOXP3 + T cells expressing surface markers related to Tregs combined from nine independent experiments (three donors). Expression of CTLA-4 was measured by intracellular staining. ( B ) Percentage (mean ± SEM) of CD8 + CD25 + T cells expressing cytokines related to Tregs measured by intracellular staining and flow cytometry. Data are combined from nine independent experiments (three donors). ( C ) During suboptimal stimulation with SEC1, the culture supernatant was collected every 2 d and analyzed using a Milliplex Luminex cytokine analysis kit. Data shown are combined results from six independent experiments (two donors).

Techniques Used: Expressing, Concentration Assay, Flow Cytometry, Cytometry, Luminex, Enzyme-linked Immunosorbent Assay, Staining

Time-dependent expression kinetics of FOXP3 in CD8 + CD25 + T cells induced by stimulation with SEC1. Human PBMCs were stimulated under optimal (1 μg/ml) and suboptimal (1 ng/ml) concentrations of SEC1 for up to 8 d. Expression of CD25 and FOXP3 in CD8 T cells was measured using flow cytometry. ( A ) Data shown are a single representative experiment gated on CD8 + T cells. ( B ) Percentage (mean ± SEM) of CD8 + CD25 + FOXP3 + T cells combined from nine independent experiments (three donors).
Figure Legend Snippet: Time-dependent expression kinetics of FOXP3 in CD8 + CD25 + T cells induced by stimulation with SEC1. Human PBMCs were stimulated under optimal (1 μg/ml) and suboptimal (1 ng/ml) concentrations of SEC1 for up to 8 d. Expression of CD25 and FOXP3 in CD8 T cells was measured using flow cytometry. ( A ) Data shown are a single representative experiment gated on CD8 + T cells. ( B ) Percentage (mean ± SEM) of CD8 + CD25 + FOXP3 + T cells combined from nine independent experiments (three donors).

Techniques Used: Expressing, Flow Cytometry, Cytometry

Concentration-dependent expression kinetics of FOXP3 in CD8 + CD25 + T cells by SEC1. ( A ) Human T cell proliferation after exposure to SEC1 for 4 d was measured by the incorporation of [ 3 H]thymidine. The data shown are the mean ± SEM of nine independent experiments (three separate experiments for each of three different donors). ( C ) Human PBMCs were stimulated with various concentrations of SEC1 for 4 d, and expression of CD25 and FOXP3 in CD8 + T cells was measured by flow cytometry. The data shown are from a single representative experiment. ( B ) Percentage (mean ± SEM) of CD8 + CD25 + FOXP3 + T cells combined from nine independent experiments.
Figure Legend Snippet: Concentration-dependent expression kinetics of FOXP3 in CD8 + CD25 + T cells by SEC1. ( A ) Human T cell proliferation after exposure to SEC1 for 4 d was measured by the incorporation of [ 3 H]thymidine. The data shown are the mean ± SEM of nine independent experiments (three separate experiments for each of three different donors). ( C ) Human PBMCs were stimulated with various concentrations of SEC1 for 4 d, and expression of CD25 and FOXP3 in CD8 + T cells was measured by flow cytometry. The data shown are from a single representative experiment. ( B ) Percentage (mean ± SEM) of CD8 + CD25 + FOXP3 + T cells combined from nine independent experiments.

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

Expression of galectin-1 and granzymes by CD8 + CD25 + T cells induced by optimal and suboptimal stimulation with SEC1. ( A ) PBMC/CD25 − T cells were stimulated with an optimal (1 μg/ml) and suboptimal (1 ng/ml) concentration of SEC1 for 4 and 6 d, respectively. Intracellular and surface expression of galectin-1 and intracellular expression of granzyme A and B were analyzed by flow cytometry. Data shown are representative dot plots gated on CD8 + CD25 + T cells. ( B ) Percentage (mean ± SEM) of CD8 + CD25 + FOXP3 + T cells expressing galectin-1, granzyme A, or granzyme B combined from nine independent experiments (three donors).
Figure Legend Snippet: Expression of galectin-1 and granzymes by CD8 + CD25 + T cells induced by optimal and suboptimal stimulation with SEC1. ( A ) PBMC/CD25 − T cells were stimulated with an optimal (1 μg/ml) and suboptimal (1 ng/ml) concentration of SEC1 for 4 and 6 d, respectively. Intracellular and surface expression of galectin-1 and intracellular expression of granzyme A and B were analyzed by flow cytometry. Data shown are representative dot plots gated on CD8 + CD25 + T cells. ( B ) Percentage (mean ± SEM) of CD8 + CD25 + FOXP3 + T cells expressing galectin-1, granzyme A, or granzyme B combined from nine independent experiments (three donors).

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

38) Product Images from "Tumor-derived microRNAs induce myeloid suppressor cells and predict immunotherapy resistance in melanoma"

Article Title: Tumor-derived microRNAs induce myeloid suppressor cells and predict immunotherapy resistance in melanoma

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI98060

miR regulation in EV-MDSCs. ( A ) Volcano plot of the miRs regulated in EV-MDSCs ( n = 5 HD) compared with untreated monocytes based on microarray results, identification strategy of MDSC-miRs, and relative expression of selected miRs in EV-MDSCs compared with untreated monocytes assessed by qPCR in a representative HD. ( B ) Expression of MDSC-miRs in f1 and f2 plasma EVs of melanoma patients ( n = 16). Box and whiskers (Tukey’s test). ( C ) Monocytes from HD transfected with MDSC-miR mimics (MmiRs) modulate HLA-DRA , IL6 , and CCL2 gene expression compared with monocytes treated with scrambled control used as calibrator. ( D ) Immunosuppressive activity of mono+MmiRs on autologous activated CFSE-labeled T cells, as evaluated by CD25 expression and proliferation (percentage is indicated, left), and release of IFN-γ and TNF-α (right). ( E ) Loss of immunosuppressive activity of monocytes from a melanoma patient transfected with miR inhibitors (ImiRs) prior to coincubation with autologous activated CFSE-labeled T cells, as evaluated by flow cytometry (left) and cytokine release (right). ( F ) Expression of MDSC-miRs in CD14 + cells isolated from PBMCs of melanoma patients ( n = 31) and HD ( n = 15). AU, arbitrary units. P
Figure Legend Snippet: miR regulation in EV-MDSCs. ( A ) Volcano plot of the miRs regulated in EV-MDSCs ( n = 5 HD) compared with untreated monocytes based on microarray results, identification strategy of MDSC-miRs, and relative expression of selected miRs in EV-MDSCs compared with untreated monocytes assessed by qPCR in a representative HD. ( B ) Expression of MDSC-miRs in f1 and f2 plasma EVs of melanoma patients ( n = 16). Box and whiskers (Tukey’s test). ( C ) Monocytes from HD transfected with MDSC-miR mimics (MmiRs) modulate HLA-DRA , IL6 , and CCL2 gene expression compared with monocytes treated with scrambled control used as calibrator. ( D ) Immunosuppressive activity of mono+MmiRs on autologous activated CFSE-labeled T cells, as evaluated by CD25 expression and proliferation (percentage is indicated, left), and release of IFN-γ and TNF-α (right). ( E ) Loss of immunosuppressive activity of monocytes from a melanoma patient transfected with miR inhibitors (ImiRs) prior to coincubation with autologous activated CFSE-labeled T cells, as evaluated by flow cytometry (left) and cytokine release (right). ( F ) Expression of MDSC-miRs in CD14 + cells isolated from PBMCs of melanoma patients ( n = 31) and HD ( n = 15). AU, arbitrary units. P

Techniques Used: Microarray, Expressing, Real-time Polymerase Chain Reaction, Transfection, Activity Assay, Labeling, Flow Cytometry, Cytometry, Isolation

Conversion of myeloid cells into MDSCs by melanoma EVs. ( A ) HD-CD14 + cells (Mono) incubated 24 hours with melanoma EVs (Me EVs) downregulate HLA-DR (left, representative plot and summary of n = 7 HD), increase production of cytochemokines (middle), modulate HLA-DRA , IL6 , and CCL2 gene transcription (right), and ( B ) suppress proliferation of activated CFSE-labeled T cells (percentage proliferation indicated). ( C ) CD14 + HLA-DR neg cell frequency and HLA-DR expression on gated CD14 + cells in PBMCs of melanoma patients (Pts, n = 31) and HD ( n = 15) by flow cytometry. ( D ) HLA-DRA downregulation of HD ( n = 5) and patients’ ( n = 4) monocytes cultured with melanoma EVs. ( E ) Induction of EV-MDSCs in CD14 + cells from a patient by autologous melanoma cell line EVs (left); suppressive activity on activated CD25 + T cells (percentages indicated, right). ( F ) NTA evaluation of EV size in plasma samples of patients and HD ( n = 27/group) (top); correlation of EV mean size and frequency of CD14 + HLA-DR neg in gated CD14 + cells of melanoma patients (bottom). ( G ) EV-MDSC converting potential of f1 and f2 plasma EVs from patients and HD ( n = 5/group) shown as HLA-DRA downregulation in monocytes from 2 different HD; control: melanoma EVs. Data are presented as mean ± SEM. ( H ) Autologous (auto) plasma EVs f1 and f2 convert melanoma patient’s CD14 + cells, as shown by modulation of HLA-DRA , IL6 , and CCL2 transcripts (top). EV-MDSCs generated with autologous plasma EVs f1 and f2 of melanoma patient inhibit T cell proliferation (percentages indicated, bottom). ( I ) Western blot of plasma EV fractions (f1, f2) of HD and patient. gMFI, geo mean fluorescence intensity; RE, relative expression. FC was by using as calibrator untreated monocytes. P
Figure Legend Snippet: Conversion of myeloid cells into MDSCs by melanoma EVs. ( A ) HD-CD14 + cells (Mono) incubated 24 hours with melanoma EVs (Me EVs) downregulate HLA-DR (left, representative plot and summary of n = 7 HD), increase production of cytochemokines (middle), modulate HLA-DRA , IL6 , and CCL2 gene transcription (right), and ( B ) suppress proliferation of activated CFSE-labeled T cells (percentage proliferation indicated). ( C ) CD14 + HLA-DR neg cell frequency and HLA-DR expression on gated CD14 + cells in PBMCs of melanoma patients (Pts, n = 31) and HD ( n = 15) by flow cytometry. ( D ) HLA-DRA downregulation of HD ( n = 5) and patients’ ( n = 4) monocytes cultured with melanoma EVs. ( E ) Induction of EV-MDSCs in CD14 + cells from a patient by autologous melanoma cell line EVs (left); suppressive activity on activated CD25 + T cells (percentages indicated, right). ( F ) NTA evaluation of EV size in plasma samples of patients and HD ( n = 27/group) (top); correlation of EV mean size and frequency of CD14 + HLA-DR neg in gated CD14 + cells of melanoma patients (bottom). ( G ) EV-MDSC converting potential of f1 and f2 plasma EVs from patients and HD ( n = 5/group) shown as HLA-DRA downregulation in monocytes from 2 different HD; control: melanoma EVs. Data are presented as mean ± SEM. ( H ) Autologous (auto) plasma EVs f1 and f2 convert melanoma patient’s CD14 + cells, as shown by modulation of HLA-DRA , IL6 , and CCL2 transcripts (top). EV-MDSCs generated with autologous plasma EVs f1 and f2 of melanoma patient inhibit T cell proliferation (percentages indicated, bottom). ( I ) Western blot of plasma EV fractions (f1, f2) of HD and patient. gMFI, geo mean fluorescence intensity; RE, relative expression. FC was by using as calibrator untreated monocytes. P

Techniques Used: Incubation, Labeling, Expressing, Flow Cytometry, Cytometry, Cell Culture, Activity Assay, Generated, Western Blot, Fluorescence

39) Product Images from "Irradiation enhances human T cell function by up-regulating CD70 expression on antigen-presenting cells in vitro"

Article Title: Irradiation enhances human T cell function by up-regulating CD70 expression on antigen-presenting cells in vitro

Journal: Journal of immunotherapy (Hagerstown, Md. : 1997)

doi: 10.1097/CJI.0b013e318216983d

Inhibiting CD70 decreases T-cell proliferation (A) T-cell proliferation was decreased by blocking irradiation-induced CD70 expression on DCs. Three DCs derived from monocytes were differentiated by IL-4 (1000 U/ml) and GM-CSF (1000 U/ml). Anti-CD70 and isotype control (20 μg/ml) were added to the culture before DCs were matured by CD40L (2 μg/ml), LPS (1 μg/ml), and irradiation (1000 cGy). Purified MHC-mismatched CD3 + T cells were labeled with CFSE and the labeled cells were co-cultured with the DCs. FACS analysis was performed 1 day later. (B) CD70 expression induced by irradiation of mature DCs was inhibited by shRNA. Monocytes were isolated from 2 HLA-A2 + PBMC samples, differentiated to immature DCs using GM-CSF and IL-4 for 2 days, and transduced with lentiviral shRNA, an inhibitor of CD70 (non-target shRNA and 4 clones: TRCN 0000007840, TRCN 0000007841, TRCN 0000007843, and TRCN 0000011202). After 3 days, cells underwent maturation and irradiation, and FACS analysis was performed by examining CD70 expression on DCs. (C) shRNA inhibition of CD70 led to reduced IFN-γ production. The 2 DC cultures described in (B) were pulsed with MART-1 26-35 ). (D) Inhibiting CD70 decreased T-cell proliferation. The 2 treated DCs described in (B) were co-cultured with CFSE-labeled CD3 + T cells, and FACS analysis was done 5 days later. The results from 1 of 2 representative experiments are shown.
Figure Legend Snippet: Inhibiting CD70 decreases T-cell proliferation (A) T-cell proliferation was decreased by blocking irradiation-induced CD70 expression on DCs. Three DCs derived from monocytes were differentiated by IL-4 (1000 U/ml) and GM-CSF (1000 U/ml). Anti-CD70 and isotype control (20 μg/ml) were added to the culture before DCs were matured by CD40L (2 μg/ml), LPS (1 μg/ml), and irradiation (1000 cGy). Purified MHC-mismatched CD3 + T cells were labeled with CFSE and the labeled cells were co-cultured with the DCs. FACS analysis was performed 1 day later. (B) CD70 expression induced by irradiation of mature DCs was inhibited by shRNA. Monocytes were isolated from 2 HLA-A2 + PBMC samples, differentiated to immature DCs using GM-CSF and IL-4 for 2 days, and transduced with lentiviral shRNA, an inhibitor of CD70 (non-target shRNA and 4 clones: TRCN 0000007840, TRCN 0000007841, TRCN 0000007843, and TRCN 0000011202). After 3 days, cells underwent maturation and irradiation, and FACS analysis was performed by examining CD70 expression on DCs. (C) shRNA inhibition of CD70 led to reduced IFN-γ production. The 2 DC cultures described in (B) were pulsed with MART-1 26-35 ). (D) Inhibiting CD70 decreased T-cell proliferation. The 2 treated DCs described in (B) were co-cultured with CFSE-labeled CD3 + T cells, and FACS analysis was done 5 days later. The results from 1 of 2 representative experiments are shown.

Techniques Used: Blocking Assay, Irradiation, Expressing, Derivative Assay, Purification, Labeling, Cell Culture, FACS, shRNA, Isolation, Transduction, Clone Assay, Inhibition

Up-regulating CD70 on mature DCs enhanced adoptively transferred T-cell effector function and promoted T-cell proliferation (A) Up-regulation of CD70 on mature DCs. Monocytes were isolated from 2 PBMC samples by washing out non-adherent cells. Adherent cells were then cultured in medium containing 1000 U/ml of GM-CSF and 1000 U/ml of IL-4 for 5 days. On day 6, 1000 U/ml of CD40L was added. Cells were irradiated with the indicated doses after 24 hours (top row) or 5 days (bottom row). FACS analysis was carried out by analyzing CD70 expression on lin1 – , CD11c + , and PI – populations and the % of CD70 + CD83 + cells were indicated (B) Irradiation of mature DCs enhanced alloantigen-stimulated T cell proliferation. DCs were prepared as in (A) and co-cultured with CFSE-labeled CD3 + T cells derived from 3 MHC-mismatched PBMC samples. After 5 days, FACS analysis was performed by gating of CD3 + and PI – T cells. (C) CD70 expression on DCs correlated with dose of in vitro irradiation (MFI: mean fluorescence intensity). (D) Irradiation of DCs promoted IFN-γ release in adoptively transferred CD8 + T cells. Irradiated DCs were prepared from HLAA2 + PBMCs as described in (A) ). Analysis of IFN-γ release was carried out 18 hours later. The results from 1 of 3 representative experiments are shown.
Figure Legend Snippet: Up-regulating CD70 on mature DCs enhanced adoptively transferred T-cell effector function and promoted T-cell proliferation (A) Up-regulation of CD70 on mature DCs. Monocytes were isolated from 2 PBMC samples by washing out non-adherent cells. Adherent cells were then cultured in medium containing 1000 U/ml of GM-CSF and 1000 U/ml of IL-4 for 5 days. On day 6, 1000 U/ml of CD40L was added. Cells were irradiated with the indicated doses after 24 hours (top row) or 5 days (bottom row). FACS analysis was carried out by analyzing CD70 expression on lin1 – , CD11c + , and PI – populations and the % of CD70 + CD83 + cells were indicated (B) Irradiation of mature DCs enhanced alloantigen-stimulated T cell proliferation. DCs were prepared as in (A) and co-cultured with CFSE-labeled CD3 + T cells derived from 3 MHC-mismatched PBMC samples. After 5 days, FACS analysis was performed by gating of CD3 + and PI – T cells. (C) CD70 expression on DCs correlated with dose of in vitro irradiation (MFI: mean fluorescence intensity). (D) Irradiation of DCs promoted IFN-γ release in adoptively transferred CD8 + T cells. Irradiated DCs were prepared from HLAA2 + PBMCs as described in (A) ). Analysis of IFN-γ release was carried out 18 hours later. The results from 1 of 3 representative experiments are shown.

Techniques Used: Isolation, Cell Culture, Irradiation, FACS, Expressing, Labeling, Derivative Assay, In Vitro, Fluorescence

40) Product Images from "Fimepinostat, a novel dual inhibitor of HDAC and PI3K, effectively reverses HIV-1 latency ex vivo without T cell activation"

Article Title: Fimepinostat, a novel dual inhibitor of HDAC and PI3K, effectively reverses HIV-1 latency ex vivo without T cell activation

Journal: Journal of Virus Eradication

doi:

Fimepinostat showed no T cell activation in PBMCs from HIV-1-negative donors. Expression of activation marker CD69 (a) and proliferation marker Ki67 (b) on PBMCs from HIV-1-negative donors after 48-hour incubation with indicated concentrations of fimepinostat, romidepsin and negative control (DMSO at 0.01%) on the percentages of central memory (TCM) and effector memory (TEM) CD4+ T cells, respectively. Columns represent the mean. DMSO: dimethylsulphoxide; PBMC: peripheral blood mononuclear cell; TCM: central memory T cell; TEM: effector memory T cell
Figure Legend Snippet: Fimepinostat showed no T cell activation in PBMCs from HIV-1-negative donors. Expression of activation marker CD69 (a) and proliferation marker Ki67 (b) on PBMCs from HIV-1-negative donors after 48-hour incubation with indicated concentrations of fimepinostat, romidepsin and negative control (DMSO at 0.01%) on the percentages of central memory (TCM) and effector memory (TEM) CD4+ T cells, respectively. Columns represent the mean. DMSO: dimethylsulphoxide; PBMC: peripheral blood mononuclear cell; TCM: central memory T cell; TEM: effector memory T cell

Techniques Used: Activation Assay, Expressing, Marker, Incubation, Negative Control, Transmission Electron Microscopy

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Article Title: Hypoxia Potentiates Glioma-Mediated Immunosuppression
Article Snippet: .. CD14+ monocytes were purified from PBMCs by positive selection using CD14 microbeads (Miltenyi Biotech, Auburn, CA), and CD3+ T cells were purified from PBMCs by negative selection using a Pan T Cell Isolation Kit II (Miltenyi Biotech, Auburn, CA), according to the manufacturer's instructions. .. Human monocyte assays 5×105 CD14+ monocytes were incubated with 500 µL (1×106 cells/mL) of supernatant medium from gCSCs cultured in both normoxic and hypoxic conditions, as well as in neurosphere medium as controls.

Article Title: Human thymic MR1-restricted MAIT cells are innate pathogen-reactive effectors that adapt following thymic egress
Article Snippet: .. Intracellular Cytokine Staining Assay CD8+ and DN T cells from PBMC and CB were selected using negative selection of T cells then depleted of CD4+ cells by positive selection using magnetic bead separation according to the manufacturer’s instructions (Miltenyi). .. CD4-depleted T cells were added to Mtb-infected or uninfected A549 cells at ratio of 3:1 and incubated for 16 hrs.

Article Title: Characterization of Novel PI3K? Inhibitors as Potential Therapeutics for SLE and Lupus Nephritis in Pre-Clinical Studies
Article Snippet: .. T-cell cytokine release assay CD4+ CD45RA− memory T-cells (more than 95% purity) were isolated from PBMC of healthy volunteers by negative selection using the CD4+ memory cell purification kit (Miltenyi Biotech, Bergisch Gladbach, Germany). .. Purified T-cells were incubated with PI3Kδ inhibitors for 1 h, and stimulated with 5 μg/ml anti-CD3 and 2 μg/ml anti-CD28 for 5 days.

Article Title: Highly Active Microbial Phosphoantigen Induces Rapid yet Sustained MEK/Erk- and PI-3K/Akt-Mediated Signal Transduction in Anti-Tumor Human ?? T-Cells
Article Snippet: .. Magnetic cell sorting and flow cytometry analysis γδ T-cells were isolated (to above 95% purity) from PBMC by magnetic cell sorting via positive selection with a FITC-labeled anti-TCRγδ antibody (Miltenyi Biotec). .. For flow cytometry analysis (on a FACSCalibur, BD Biosciences), cells were labelled with fluorescent monoclonal antibodies: anti-CD69-PE (BD Pharmingen), anti-TcRVγ9-PC5 (Beckman Coulter) and anti-CD4-PerCP (BD Pharmingen).

Staining:

Article Title: Human thymic MR1-restricted MAIT cells are innate pathogen-reactive effectors that adapt following thymic egress
Article Snippet: .. Intracellular Cytokine Staining Assay CD8+ and DN T cells from PBMC and CB were selected using negative selection of T cells then depleted of CD4+ cells by positive selection using magnetic bead separation according to the manufacturer’s instructions (Miltenyi). .. CD4-depleted T cells were added to Mtb-infected or uninfected A549 cells at ratio of 3:1 and incubated for 16 hrs.

Article Title: Enzymatic discovery of a HER-2/neu epitope that generates cross-reactive T cells 1
Article Snippet: .. PBMC samples which stained positive for HLA-A2 were further enriched with the CD8+ T Cell Isolation Kit II (Miltenyi Biotec) and separated by AutoMACS (Miltenyi Biotec) per the manufacturer's protocol. ..

Isolation:

Article Title: Methylglyoxal-bis-guanylhydrazone inhibits osteopontin expression and differentiation in cultured human monocytes
Article Snippet: .. Monocytes were isolated from PBMCs using anti-CD14 (Miltenyi Biotech, Auburn, CA) MACS magnetic separation system. .. PBMCs or sorted monocytes were cultured in RPMI 1640 containing L-glutamine supplemented with 10% fetal bovine serum (FBS) and 1 mM sodium pyruvate.

Article Title: Characterization of Novel PI3K? Inhibitors as Potential Therapeutics for SLE and Lupus Nephritis in Pre-Clinical Studies
Article Snippet: .. T-cell cytokine release assay CD4+ CD45RA− memory T-cells (more than 95% purity) were isolated from PBMC of healthy volunteers by negative selection using the CD4+ memory cell purification kit (Miltenyi Biotech, Bergisch Gladbach, Germany). .. Purified T-cells were incubated with PI3Kδ inhibitors for 1 h, and stimulated with 5 μg/ml anti-CD3 and 2 μg/ml anti-CD28 for 5 days.

Article Title: Highly Active Microbial Phosphoantigen Induces Rapid yet Sustained MEK/Erk- and PI-3K/Akt-Mediated Signal Transduction in Anti-Tumor Human ?? T-Cells
Article Snippet: .. Magnetic cell sorting and flow cytometry analysis γδ T-cells were isolated (to above 95% purity) from PBMC by magnetic cell sorting via positive selection with a FITC-labeled anti-TCRγδ antibody (Miltenyi Biotec). .. For flow cytometry analysis (on a FACSCalibur, BD Biosciences), cells were labelled with fluorescent monoclonal antibodies: anti-CD69-PE (BD Pharmingen), anti-TcRVγ9-PC5 (Beckman Coulter) and anti-CD4-PerCP (BD Pharmingen).

Cytometry:

Article Title: Highly Active Microbial Phosphoantigen Induces Rapid yet Sustained MEK/Erk- and PI-3K/Akt-Mediated Signal Transduction in Anti-Tumor Human ?? T-Cells
Article Snippet: .. Magnetic cell sorting and flow cytometry analysis γδ T-cells were isolated (to above 95% purity) from PBMC by magnetic cell sorting via positive selection with a FITC-labeled anti-TCRγδ antibody (Miltenyi Biotec). .. For flow cytometry analysis (on a FACSCalibur, BD Biosciences), cells were labelled with fluorescent monoclonal antibodies: anti-CD69-PE (BD Pharmingen), anti-TcRVγ9-PC5 (Beckman Coulter) and anti-CD4-PerCP (BD Pharmingen).

Purification:

Article Title: Hypoxia Potentiates Glioma-Mediated Immunosuppression
Article Snippet: .. CD14+ monocytes were purified from PBMCs by positive selection using CD14 microbeads (Miltenyi Biotech, Auburn, CA), and CD3+ T cells were purified from PBMCs by negative selection using a Pan T Cell Isolation Kit II (Miltenyi Biotech, Auburn, CA), according to the manufacturer's instructions. .. Human monocyte assays 5×105 CD14+ monocytes were incubated with 500 µL (1×106 cells/mL) of supernatant medium from gCSCs cultured in both normoxic and hypoxic conditions, as well as in neurosphere medium as controls.

Article Title: Characterization of Novel PI3K? Inhibitors as Potential Therapeutics for SLE and Lupus Nephritis in Pre-Clinical Studies
Article Snippet: .. T-cell cytokine release assay CD4+ CD45RA− memory T-cells (more than 95% purity) were isolated from PBMC of healthy volunteers by negative selection using the CD4+ memory cell purification kit (Miltenyi Biotech, Bergisch Gladbach, Germany). .. Purified T-cells were incubated with PI3Kδ inhibitors for 1 h, and stimulated with 5 μg/ml anti-CD3 and 2 μg/ml anti-CD28 for 5 days.

Incubation:

Article Title: NS1 Specific CD8+ T-Cells with Effector Function and TRBV11 Dominance in a Patient with Parvovirus B19 Associated Inflammatory Cardiomyopathy
Article Snippet: .. SALK- and GLCP-reactive cells were enriched from PBMCs after incubation with these two peptides over 6 h using the IFNγ secretion assay according to the manufacturer's instructions (Miltenyi Biotech, Bergisch Gladbach, Germany). .. PBMCs were separated from peripheral blood obtained in Vacutainer® EDTA tubes (BD Biosciences, Heidelberg, Germany) using the LSM 1077 Lymphocyte Separation Medium (PAA, Pasching, Germany) density gradient.

Release Assay:

Article Title: Characterization of Novel PI3K? Inhibitors as Potential Therapeutics for SLE and Lupus Nephritis in Pre-Clinical Studies
Article Snippet: .. T-cell cytokine release assay CD4+ CD45RA− memory T-cells (more than 95% purity) were isolated from PBMC of healthy volunteers by negative selection using the CD4+ memory cell purification kit (Miltenyi Biotech, Bergisch Gladbach, Germany). .. Purified T-cells were incubated with PI3Kδ inhibitors for 1 h, and stimulated with 5 μg/ml anti-CD3 and 2 μg/ml anti-CD28 for 5 days.

FACS:

Article Title: Highly Active Microbial Phosphoantigen Induces Rapid yet Sustained MEK/Erk- and PI-3K/Akt-Mediated Signal Transduction in Anti-Tumor Human ?? T-Cells
Article Snippet: .. Magnetic cell sorting and flow cytometry analysis γδ T-cells were isolated (to above 95% purity) from PBMC by magnetic cell sorting via positive selection with a FITC-labeled anti-TCRγδ antibody (Miltenyi Biotec). .. For flow cytometry analysis (on a FACSCalibur, BD Biosciences), cells were labelled with fluorescent monoclonal antibodies: anti-CD69-PE (BD Pharmingen), anti-TcRVγ9-PC5 (Beckman Coulter) and anti-CD4-PerCP (BD Pharmingen).

Magnetic Cell Separation:

Article Title: Methylglyoxal-bis-guanylhydrazone inhibits osteopontin expression and differentiation in cultured human monocytes
Article Snippet: .. Monocytes were isolated from PBMCs using anti-CD14 (Miltenyi Biotech, Auburn, CA) MACS magnetic separation system. .. PBMCs or sorted monocytes were cultured in RPMI 1640 containing L-glutamine supplemented with 10% fetal bovine serum (FBS) and 1 mM sodium pyruvate.

Flow Cytometry:

Article Title: Highly Active Microbial Phosphoantigen Induces Rapid yet Sustained MEK/Erk- and PI-3K/Akt-Mediated Signal Transduction in Anti-Tumor Human ?? T-Cells
Article Snippet: .. Magnetic cell sorting and flow cytometry analysis γδ T-cells were isolated (to above 95% purity) from PBMC by magnetic cell sorting via positive selection with a FITC-labeled anti-TCRγδ antibody (Miltenyi Biotec). .. For flow cytometry analysis (on a FACSCalibur, BD Biosciences), cells were labelled with fluorescent monoclonal antibodies: anti-CD69-PE (BD Pharmingen), anti-TcRVγ9-PC5 (Beckman Coulter) and anti-CD4-PerCP (BD Pharmingen).

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  • 95
    Miltenyi Biotec pbmcs
    SPM reversed the MGBG inhibitory effects on sOPN and CD16 expression in monocytes. <t>PBMCs</t> were cultured for 3 days with or without treatment. Cells were cultured with 10 μM MGBG and various concentrations of SPM. (A). MGBG significantly decreased sOPN; SPM reversed the MGBG inhibitory effects on sOPN. The average sOPN level of 3 day untreated and 10 μM MGBG treated cells was 30 and 2 ng/ml, respectively. n = 6, means and SEM. (B). MGBG inhibited monocyte CD16 expression; SPM reversed the MGBG inhibitory effects on CD16 expression. Cultured PBMCs were double stained with <t>CD14</t> and CD16 antibodies for flow cytometry analysis. The average CD16 geometric mean in untreated and MGBG treated CD14+ monocytes were measured at 510 and 249, respectively. n = 3, means and SEM. Data was presented as a percentage of inhibition of MGMG treatment only.
    Pbmcs, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 95/100, based on 3301 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Miltenyi Biotec pha pbmcs
    AZT and ddI susceptibility of HIV-1 subtype E virus isolates from a family. (A) Replication kinetics. <t>PHA-PBMCs</t> were infected with virus stock (100 TCID 50 ) in the culture medium was monitored at the indicated time points. (B) Dose-response curve. Infected cells were cultured in the indicated concentrations of AZT (left panel) or ddI (right panel). RT activity at the peak of infection is expressed as a percentage of that of the untreated culture. Mean values with the standard errors of the three independent experiments for AZT and mean values of quadruplicates of an experiment for ddI are plotted.
    Pha Pbmcs, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pha pbmcs/product/Miltenyi Biotec
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    85
    Miltenyi Biotec lr eoc pbmcs
    Proliferation of Vγ9Vδ2 <t>PBMCs</t> from <t>EOC</t> patients and from healthy female donors following BrHPP or zoledronate stimulation. Vδ2 + CD3 + cell frequencies among expanded cells ( A ) and Vδ2 + CD3 + cell numbers generated from 1×10 6 PBMCs ( B ) were measured at 14 days after BrHPP ( ▴ ) or zoledronate (Zol) ( ▪ ) stimulation. The results from n = 60 patient PBMCs and n = 13 donor PBMCs are shown.
    Lr Eoc Pbmcs, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SPM reversed the MGBG inhibitory effects on sOPN and CD16 expression in monocytes. PBMCs were cultured for 3 days with or without treatment. Cells were cultured with 10 μM MGBG and various concentrations of SPM. (A). MGBG significantly decreased sOPN; SPM reversed the MGBG inhibitory effects on sOPN. The average sOPN level of 3 day untreated and 10 μM MGBG treated cells was 30 and 2 ng/ml, respectively. n = 6, means and SEM. (B). MGBG inhibited monocyte CD16 expression; SPM reversed the MGBG inhibitory effects on CD16 expression. Cultured PBMCs were double stained with CD14 and CD16 antibodies for flow cytometry analysis. The average CD16 geometric mean in untreated and MGBG treated CD14+ monocytes were measured at 510 and 249, respectively. n = 3, means and SEM. Data was presented as a percentage of inhibition of MGMG treatment only.

    Journal: PLoS ONE

    Article Title: Methylglyoxal-bis-guanylhydrazone inhibits osteopontin expression and differentiation in cultured human monocytes

    doi: 10.1371/journal.pone.0192680

    Figure Lengend Snippet: SPM reversed the MGBG inhibitory effects on sOPN and CD16 expression in monocytes. PBMCs were cultured for 3 days with or without treatment. Cells were cultured with 10 μM MGBG and various concentrations of SPM. (A). MGBG significantly decreased sOPN; SPM reversed the MGBG inhibitory effects on sOPN. The average sOPN level of 3 day untreated and 10 μM MGBG treated cells was 30 and 2 ng/ml, respectively. n = 6, means and SEM. (B). MGBG inhibited monocyte CD16 expression; SPM reversed the MGBG inhibitory effects on CD16 expression. Cultured PBMCs were double stained with CD14 and CD16 antibodies for flow cytometry analysis. The average CD16 geometric mean in untreated and MGBG treated CD14+ monocytes were measured at 510 and 249, respectively. n = 3, means and SEM. Data was presented as a percentage of inhibition of MGMG treatment only.

    Article Snippet: Monocytes were isolated from PBMCs using anti-CD14 (Miltenyi Biotech, Auburn, CA) MACS magnetic separation system.

    Techniques: Expressing, Cell Culture, Staining, Flow Cytometry, Cytometry, Inhibition

    MGBG showed greater inhibition on CD16 expression in monocytes than that in differentiated macrophages. Monocytes were isolated from PBMCs using CD14 microbeads. Cells were cultured for 1, 3, and 6 days with various concentrations of MGBG, and collected for CD16 expression measurement using flow cytometry. MGBG showed greater inhibitory effect on CD 16 expression in 1 and 3 day monocytes than that in differentiated macrophages. The average CD16 geometric mean fluorescence was measured at 108, 448, and 249 for 1, 3, and 6 day untreated cells, respectively. n = 4, means and SEM.

    Journal: PLoS ONE

    Article Title: Methylglyoxal-bis-guanylhydrazone inhibits osteopontin expression and differentiation in cultured human monocytes

    doi: 10.1371/journal.pone.0192680

    Figure Lengend Snippet: MGBG showed greater inhibition on CD16 expression in monocytes than that in differentiated macrophages. Monocytes were isolated from PBMCs using CD14 microbeads. Cells were cultured for 1, 3, and 6 days with various concentrations of MGBG, and collected for CD16 expression measurement using flow cytometry. MGBG showed greater inhibitory effect on CD 16 expression in 1 and 3 day monocytes than that in differentiated macrophages. The average CD16 geometric mean fluorescence was measured at 108, 448, and 249 for 1, 3, and 6 day untreated cells, respectively. n = 4, means and SEM.

    Article Snippet: Monocytes were isolated from PBMCs using anti-CD14 (Miltenyi Biotech, Auburn, CA) MACS magnetic separation system.

    Techniques: Inhibition, Expressing, Isolation, Cell Culture, Flow Cytometry, Cytometry, Fluorescence

    HER-2/neu p373–382-specific CD8 + T cells have increased recognition of human breast cancer cell lines (A–B) Shown are the mean (±SEM, n=3) numbers of ELISpots obtained in response of pFLU, p369–377, or p373–382 peptide-specific CD8 + T cells derived from short-term culture to (A) autologous PBMC pulsed with pFLU, p369–377, or p373–382 or (B) irradiated tumor cells. Results are from a single donor and are representative of similar results obtained with 3 other donors. (C) Shown are the mean (±SEM, n=4) % lysis values calculated from the impedance-based lysis assay at 10 hours following the addition of T cells. Also shown are the expression levels of HER-2/neu and HLA-A2 genotyping results for each of the cell lines. Results are pertinent to panels B and C. HER-2/neu overexpression levels in each cell line and HLA-A2 expression are indicated below the x-axis. Expression levels were determined by flow cytometry and RT-PCR. p-values in panels B and C were calculated using an unpaired Student's t-test. (D) Shown are the mean (±SEM, n=3) numbers of IFN-γ ELISpots obtained in response to pFLU or p373–382 peptide-specific CD8 + T cells derived from short-term culture to autologous PBMC pulsed with pFLU, p373–382, HER-2/neu ECD or ovalbumin protein. Experiment was repeated twice in triplicate with both experiments yielding similar results.

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

    Article Title: Enzymatic discovery of a HER-2/neu epitope that generates cross-reactive T cells 1

    doi: 10.4049/jimmunol.1201264

    Figure Lengend Snippet: HER-2/neu p373–382-specific CD8 + T cells have increased recognition of human breast cancer cell lines (A–B) Shown are the mean (±SEM, n=3) numbers of ELISpots obtained in response of pFLU, p369–377, or p373–382 peptide-specific CD8 + T cells derived from short-term culture to (A) autologous PBMC pulsed with pFLU, p369–377, or p373–382 or (B) irradiated tumor cells. Results are from a single donor and are representative of similar results obtained with 3 other donors. (C) Shown are the mean (±SEM, n=4) % lysis values calculated from the impedance-based lysis assay at 10 hours following the addition of T cells. Also shown are the expression levels of HER-2/neu and HLA-A2 genotyping results for each of the cell lines. Results are pertinent to panels B and C. HER-2/neu overexpression levels in each cell line and HLA-A2 expression are indicated below the x-axis. Expression levels were determined by flow cytometry and RT-PCR. p-values in panels B and C were calculated using an unpaired Student's t-test. (D) Shown are the mean (±SEM, n=3) numbers of IFN-γ ELISpots obtained in response to pFLU or p373–382 peptide-specific CD8 + T cells derived from short-term culture to autologous PBMC pulsed with pFLU, p373–382, HER-2/neu ECD or ovalbumin protein. Experiment was repeated twice in triplicate with both experiments yielding similar results.

    Article Snippet: PBMC samples which stained positive for HLA-A2 were further enriched with the CD8+ T Cell Isolation Kit II (Miltenyi Biotec) and separated by AutoMACS (Miltenyi Biotec) per the manufacturer's protocol.

    Techniques: Derivative Assay, Irradiation, Lysis, Expressing, Over Expression, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction

    IFN-γ and lytic responses of p373–382-generated T cells are HLA class I restricted (A–B) Shown are the mean (±SEM, n=3) numbers of p373–382-specific ELISpots obtained in response to p369–377 or p373–382 peptide-specific CD8 + T cells derived from short-term culture to autologous PBMC pulsed with p369–377 or p373–382 in the presence or absence of (A) HLA-ABC blocking antibody or (B) HLA-A2 blocking antibody. One representative experiment of three is shown. (C) Shown are the mean (±SEM, n=4) % lysis values calculated from the impedance-based lysis assay at 10 hours following the addition of T cells. The target cells were pretreated with or without 10 μg/ml HLA-ABC blocking antibody or HLA-A2 blocking antibody and appropriate isotype control antibodies 1 hour prior to T cell addition. Similar results were obtained with T cells generated from 3 other donors. p-values were calculated using a non-parametric Mann-Whitney Test.

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

    Article Title: Enzymatic discovery of a HER-2/neu epitope that generates cross-reactive T cells 1

    doi: 10.4049/jimmunol.1201264

    Figure Lengend Snippet: IFN-γ and lytic responses of p373–382-generated T cells are HLA class I restricted (A–B) Shown are the mean (±SEM, n=3) numbers of p373–382-specific ELISpots obtained in response to p369–377 or p373–382 peptide-specific CD8 + T cells derived from short-term culture to autologous PBMC pulsed with p369–377 or p373–382 in the presence or absence of (A) HLA-ABC blocking antibody or (B) HLA-A2 blocking antibody. One representative experiment of three is shown. (C) Shown are the mean (±SEM, n=4) % lysis values calculated from the impedance-based lysis assay at 10 hours following the addition of T cells. The target cells were pretreated with or without 10 μg/ml HLA-ABC blocking antibody or HLA-A2 blocking antibody and appropriate isotype control antibodies 1 hour prior to T cell addition. Similar results were obtained with T cells generated from 3 other donors. p-values were calculated using a non-parametric Mann-Whitney Test.

    Article Snippet: PBMC samples which stained positive for HLA-A2 were further enriched with the CD8+ T Cell Isolation Kit II (Miltenyi Biotec) and separated by AutoMACS (Miltenyi Biotec) per the manufacturer's protocol.

    Techniques: Generated, Derivative Assay, Blocking Assay, Lysis, MANN-WHITNEY

    AZT and ddI susceptibility of HIV-1 subtype E virus isolates from a family. (A) Replication kinetics. PHA-PBMCs were infected with virus stock (100 TCID 50 ) in the culture medium was monitored at the indicated time points. (B) Dose-response curve. Infected cells were cultured in the indicated concentrations of AZT (left panel) or ddI (right panel). RT activity at the peak of infection is expressed as a percentage of that of the untreated culture. Mean values with the standard errors of the three independent experiments for AZT and mean values of quadruplicates of an experiment for ddI are plotted.

    Journal: Journal of Virology

    Article Title: Convergent Evolution of Reverse Transcriptase (RT) Genes of Human Immunodeficiency Virus Type 1 Subtypes E and B following Nucleoside Analogue RT Inhibitor Therapies

    doi:

    Figure Lengend Snippet: AZT and ddI susceptibility of HIV-1 subtype E virus isolates from a family. (A) Replication kinetics. PHA-PBMCs were infected with virus stock (100 TCID 50 ) in the culture medium was monitored at the indicated time points. (B) Dose-response curve. Infected cells were cultured in the indicated concentrations of AZT (left panel) or ddI (right panel). RT activity at the peak of infection is expressed as a percentage of that of the untreated culture. Mean values with the standard errors of the three independent experiments for AZT and mean values of quadruplicates of an experiment for ddI are plotted.

    Article Snippet: Because HIV-1NH2 and HIV-1NH3 replicated poorly in the PHA-PBMCs, PBMCs were depleted of CD8+ cells with anti-CD8 antibody and a magnetic cell sorter (Miltenyi Biotec, Bergisch-Gladbach, Germany), and the CD8-positive-cell-depleted PHA-PBMCs were used for the present assay.

    Techniques: Infection, Cell Culture, Activity Assay

    Proliferation of Vγ9Vδ2 PBMCs from EOC patients and from healthy female donors following BrHPP or zoledronate stimulation. Vδ2 + CD3 + cell frequencies among expanded cells ( A ) and Vδ2 + CD3 + cell numbers generated from 1×10 6 PBMCs ( B ) were measured at 14 days after BrHPP ( ▴ ) or zoledronate (Zol) ( ▪ ) stimulation. The results from n = 60 patient PBMCs and n = 13 donor PBMCs are shown.

    Journal: PLoS ONE

    Article Title: A Quantitative Deficiency in Peripheral Blood V?9V?2 Cells Is a Negative Prognostic Biomarker in Ovarian Cancer Patients

    doi: 10.1371/journal.pone.0063322

    Figure Lengend Snippet: Proliferation of Vγ9Vδ2 PBMCs from EOC patients and from healthy female donors following BrHPP or zoledronate stimulation. Vδ2 + CD3 + cell frequencies among expanded cells ( A ) and Vδ2 + CD3 + cell numbers generated from 1×10 6 PBMCs ( B ) were measured at 14 days after BrHPP ( ▴ ) or zoledronate (Zol) ( ▪ ) stimulation. The results from n = 60 patient PBMCs and n = 13 donor PBMCs are shown.

    Article Snippet: In additional assays, we tested an oblique method of cell depletion to remove Tregs from LR EOC PBMCs while preserving CD25+ activated cells with the use of the CD4+ CD25+ CD127dim/− Regulatory T Cell Isolation Kit II from Miltenyi Biotec (data not shown).

    Techniques: Generated

    Disease-free survival of chemotherapy-treated EOC patients according to Vγ9Vδ2 cell frequencies among ex vivo PBMCs. Disease-free survival Kaplan-Meier curves of patients with Vγ9Vδ2 cell frequencies in PBMCs of 0.35% or less (≤0.35%) (n = 9) or greater than 0.35% ( > 0.35%) (n = 43) at the time of blood collection. p value

    Journal: PLoS ONE

    Article Title: A Quantitative Deficiency in Peripheral Blood V?9V?2 Cells Is a Negative Prognostic Biomarker in Ovarian Cancer Patients

    doi: 10.1371/journal.pone.0063322

    Figure Lengend Snippet: Disease-free survival of chemotherapy-treated EOC patients according to Vγ9Vδ2 cell frequencies among ex vivo PBMCs. Disease-free survival Kaplan-Meier curves of patients with Vγ9Vδ2 cell frequencies in PBMCs of 0.35% or less (≤0.35%) (n = 9) or greater than 0.35% ( > 0.35%) (n = 43) at the time of blood collection. p value

    Article Snippet: In additional assays, we tested an oblique method of cell depletion to remove Tregs from LR EOC PBMCs while preserving CD25+ activated cells with the use of the CD4+ CD25+ CD127dim/− Regulatory T Cell Isolation Kit II from Miltenyi Biotec (data not shown).

    Techniques: Ex Vivo

    A reduced naive Vγ9Vδ2 subset in ex vivo LR EOC PBMCs. Percentages of naive (CD27 + CD45RA + ), central memory (CM) (CD27 + CD45RA − ), effector memory (EM) (CD27 − CD45RA − ) and terminally differentiated effector memory (TEMRA) (CD27 − CD45RA + ) cells among the Vδ2 + CD3 + cells in ex vivo LR (n = 13) and R (n = 11) EOC PBMCs.

    Journal: PLoS ONE

    Article Title: A Quantitative Deficiency in Peripheral Blood V?9V?2 Cells Is a Negative Prognostic Biomarker in Ovarian Cancer Patients

    doi: 10.1371/journal.pone.0063322

    Figure Lengend Snippet: A reduced naive Vγ9Vδ2 subset in ex vivo LR EOC PBMCs. Percentages of naive (CD27 + CD45RA + ), central memory (CM) (CD27 + CD45RA − ), effector memory (EM) (CD27 − CD45RA − ) and terminally differentiated effector memory (TEMRA) (CD27 − CD45RA + ) cells among the Vδ2 + CD3 + cells in ex vivo LR (n = 13) and R (n = 11) EOC PBMCs.

    Article Snippet: In additional assays, we tested an oblique method of cell depletion to remove Tregs from LR EOC PBMCs while preserving CD25+ activated cells with the use of the CD4+ CD25+ CD127dim/− Regulatory T Cell Isolation Kit II from Miltenyi Biotec (data not shown).

    Techniques: Ex Vivo

    Reduced functionality of Vγ9Vδ2 cells in LR EOC PBMCs without defects in expression level of CD3ζ and CRTAM molecules. A, B ) Vδ2 + CD3 + cell fold increases in LR (n = 16) and R (n = 32) EOC PBMCs at 14 days after BrHPP ( A ) or Zol ( B ) stimulation. C ) Percentages of IFN-γ + cells in the Vδ2 + CD3 + cells measured at 5 h after the stimulation of ex vivo LR (n = 10) and R (n = 10) PBMCs with BrHPP (3 µM) or PMA/ionomycin (PMA/iono). D ) Percentages of IFN-γ + cells ( left panel ) and TNF-α + cells ( right panel ) among the Vδ2 + CD3 + cells measured at 5 h after stimulations of ex vivo LR (n = 4) and R (n = 4) PBMCs with increasing doses of BrHPP (0.1 to 6 µM). E ) Expression of the CD3ζ chain measured on Vγ9Vδ2 PBMCs from the LR and R groups. Percentage ( upper panel ) and MFI ( lower panel ) of CD3ζ staining in the Vδ2 + CD3 + cells from LR EOC PBMCs (n = 5), R EOC PBMCs (n = 3) and R donor PBMCs (n = 3). F ) CRTAM expression on Vγ9Vδ2 cells measured at 20 h after stimulation of LR (n = 8) and R (n = 8) EOC PBMCs with BrHPP. No stim: no stimulation.

    Journal: PLoS ONE

    Article Title: A Quantitative Deficiency in Peripheral Blood V?9V?2 Cells Is a Negative Prognostic Biomarker in Ovarian Cancer Patients

    doi: 10.1371/journal.pone.0063322

    Figure Lengend Snippet: Reduced functionality of Vγ9Vδ2 cells in LR EOC PBMCs without defects in expression level of CD3ζ and CRTAM molecules. A, B ) Vδ2 + CD3 + cell fold increases in LR (n = 16) and R (n = 32) EOC PBMCs at 14 days after BrHPP ( A ) or Zol ( B ) stimulation. C ) Percentages of IFN-γ + cells in the Vδ2 + CD3 + cells measured at 5 h after the stimulation of ex vivo LR (n = 10) and R (n = 10) PBMCs with BrHPP (3 µM) or PMA/ionomycin (PMA/iono). D ) Percentages of IFN-γ + cells ( left panel ) and TNF-α + cells ( right panel ) among the Vδ2 + CD3 + cells measured at 5 h after stimulations of ex vivo LR (n = 4) and R (n = 4) PBMCs with increasing doses of BrHPP (0.1 to 6 µM). E ) Expression of the CD3ζ chain measured on Vγ9Vδ2 PBMCs from the LR and R groups. Percentage ( upper panel ) and MFI ( lower panel ) of CD3ζ staining in the Vδ2 + CD3 + cells from LR EOC PBMCs (n = 5), R EOC PBMCs (n = 3) and R donor PBMCs (n = 3). F ) CRTAM expression on Vγ9Vδ2 cells measured at 20 h after stimulation of LR (n = 8) and R (n = 8) EOC PBMCs with BrHPP. No stim: no stimulation.

    Article Snippet: In additional assays, we tested an oblique method of cell depletion to remove Tregs from LR EOC PBMCs while preserving CD25+ activated cells with the use of the CD4+ CD25+ CD127dim/− Regulatory T Cell Isolation Kit II from Miltenyi Biotec (data not shown).

    Techniques: Expressing, Ex Vivo, Staining

    An imbalanced ratio of Tregs and Vγ9Vδ2 cells in ex vivo LR EOC PBMCs. A ) CD4 + CD25 high FoxP3 high cell (Treg) frequencies and corresponding Vδ2 + CD3 + cell frequencies in ex vivo LR (n = 13) and R (n = 11) PBMCs. B ) The ratios of Tregs (%) to Vδ2 + CD3 + cells (%) (Treg:Vδ2 + ratio) among PBMCs in the LR and R groups. C ) LR PBMCs from EOC patients (n = 6) were stimulated with BrHPP (▴) or Zol (▪) and IL-2 before and after the depletion of CD25 + cells; the proliferation of Vδ2 + CD3 + cells was analyzed on day 7. Vδ2 + CD3 + cell frequencies among expanded cells ( left panel ) and Vδ2 + CD3 + cell fold increases ( right panel ) are shown.

    Journal: PLoS ONE

    Article Title: A Quantitative Deficiency in Peripheral Blood V?9V?2 Cells Is a Negative Prognostic Biomarker in Ovarian Cancer Patients

    doi: 10.1371/journal.pone.0063322

    Figure Lengend Snippet: An imbalanced ratio of Tregs and Vγ9Vδ2 cells in ex vivo LR EOC PBMCs. A ) CD4 + CD25 high FoxP3 high cell (Treg) frequencies and corresponding Vδ2 + CD3 + cell frequencies in ex vivo LR (n = 13) and R (n = 11) PBMCs. B ) The ratios of Tregs (%) to Vδ2 + CD3 + cells (%) (Treg:Vδ2 + ratio) among PBMCs in the LR and R groups. C ) LR PBMCs from EOC patients (n = 6) were stimulated with BrHPP (▴) or Zol (▪) and IL-2 before and after the depletion of CD25 + cells; the proliferation of Vδ2 + CD3 + cells was analyzed on day 7. Vδ2 + CD3 + cell frequencies among expanded cells ( left panel ) and Vδ2 + CD3 + cell fold increases ( right panel ) are shown.

    Article Snippet: In additional assays, we tested an oblique method of cell depletion to remove Tregs from LR EOC PBMCs while preserving CD25+ activated cells with the use of the CD4+ CD25+ CD127dim/− Regulatory T Cell Isolation Kit II from Miltenyi Biotec (data not shown).

    Techniques: Ex Vivo

    Specific quantitative deficiencies of Vγ9Vδ2 cells in ex vivo LR EOC PBMCs. Frequencies of Vδ2 + CD3 + cells ( A ), Vδ2 − γδ + CD3 + cells ( B ) and CD3 + cells ( C ) among LR (n = 16), Br-LR (n = 8), Zol-LR (n = 4) and R (n = 32) EOC PBMCs are shown. Differences between LR group and each of other groups are indicated. A ) A receiver-operator characteristic (ROC) analysis was performed in which LR PBMC samples were compared to the other PBMC samples. Dashed lines indicate cut-offs at 0.35% and 0.8%. Sp: Specificity. Se: sensibility.

    Journal: PLoS ONE

    Article Title: A Quantitative Deficiency in Peripheral Blood V?9V?2 Cells Is a Negative Prognostic Biomarker in Ovarian Cancer Patients

    doi: 10.1371/journal.pone.0063322

    Figure Lengend Snippet: Specific quantitative deficiencies of Vγ9Vδ2 cells in ex vivo LR EOC PBMCs. Frequencies of Vδ2 + CD3 + cells ( A ), Vδ2 − γδ + CD3 + cells ( B ) and CD3 + cells ( C ) among LR (n = 16), Br-LR (n = 8), Zol-LR (n = 4) and R (n = 32) EOC PBMCs are shown. Differences between LR group and each of other groups are indicated. A ) A receiver-operator characteristic (ROC) analysis was performed in which LR PBMC samples were compared to the other PBMC samples. Dashed lines indicate cut-offs at 0.35% and 0.8%. Sp: Specificity. Se: sensibility.

    Article Snippet: In additional assays, we tested an oblique method of cell depletion to remove Tregs from LR EOC PBMCs while preserving CD25+ activated cells with the use of the CD4+ CD25+ CD127dim/− Regulatory T Cell Isolation Kit II from Miltenyi Biotec (data not shown).

    Techniques: Ex Vivo