Structured Review

Biochrom cfse labelled pbmcs
Analysis for proliferation of CD4+ T cells after delivery of the TT-derived T-cell epitope to slanDCs via the novel immuno targeting system. (A) <t>CFSE</t> labeled <t>PBMCs</t> were prepared and incubated at 37°C for eight days with either full length TT (b) or the linker module containing (d) or lacking (c) the TT-derived T-cell epitope TT p and analysed by FACS. The data indicate that PBMCs of the selected donor contain anti-TT memory T cells that can be recalled by full length TT and to a less extent by the TT p peptide linker but not by the linker peptide lacking the TT p epitope. Untreated PBMCs (a). (B) CFSE labelled PBMCs were incubated with either the antigen-containing scaffold (c) or the single components (a,b) at 4°C. Unbound material was removed by washing.
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1) Product Images from "A Novel Modular Antigen Delivery System for Immuno Targeting of Human 6-sulfo LacNAc-Positive Blood Dendritic Cells (SlanDCs)"

Article Title: A Novel Modular Antigen Delivery System for Immuno Targeting of Human 6-sulfo LacNAc-Positive Blood Dendritic Cells (SlanDCs)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0016315

Analysis for proliferation of CD4+ T cells after delivery of the TT-derived T-cell epitope to slanDCs via the novel immuno targeting system. (A) CFSE labeled PBMCs were prepared and incubated at 37°C for eight days with either full length TT (b) or the linker module containing (d) or lacking (c) the TT-derived T-cell epitope TT p and analysed by FACS. The data indicate that PBMCs of the selected donor contain anti-TT memory T cells that can be recalled by full length TT and to a less extent by the TT p peptide linker but not by the linker peptide lacking the TT p epitope. Untreated PBMCs (a). (B) CFSE labelled PBMCs were incubated with either the antigen-containing scaffold (c) or the single components (a,b) at 4°C. Unbound material was removed by washing.
Figure Legend Snippet: Analysis for proliferation of CD4+ T cells after delivery of the TT-derived T-cell epitope to slanDCs via the novel immuno targeting system. (A) CFSE labeled PBMCs were prepared and incubated at 37°C for eight days with either full length TT (b) or the linker module containing (d) or lacking (c) the TT-derived T-cell epitope TT p and analysed by FACS. The data indicate that PBMCs of the selected donor contain anti-TT memory T cells that can be recalled by full length TT and to a less extent by the TT p peptide linker but not by the linker peptide lacking the TT p epitope. Untreated PBMCs (a). (B) CFSE labelled PBMCs were incubated with either the antigen-containing scaffold (c) or the single components (a,b) at 4°C. Unbound material was removed by washing.

Techniques Used: Derivative Assay, Labeling, Incubation, FACS

2) Product Images from "Effects of TLR agonists on maturation and function of 3-day dendritic cells from AML patients in complete remission"

Article Title: Effects of TLR agonists on maturation and function of 3-day dendritic cells from AML patients in complete remission

Journal: Journal of Translational Medicine

doi: 10.1186/1479-5876-9-151

Activation of allogeneic T cells by DCs . PBMCs from healthy unrelated donors were stained with CFSE and cocultured with DCs at a ratio of 10:1. After 6 days, proliferation was assessed by flow cytometry. (A) Representative examples of unstimulated cells and proliferating cells are shown in the presence of PHA (3 μg/mL) and after coculture with DCs generated from HCs and two AML patients (AML #5, AML #12). (B) Stimulation index of DCs generated from HCs and AML patients with cocktail C, CP, R, and RP (n = 3-9) are summarized as mean ± SD.
Figure Legend Snippet: Activation of allogeneic T cells by DCs . PBMCs from healthy unrelated donors were stained with CFSE and cocultured with DCs at a ratio of 10:1. After 6 days, proliferation was assessed by flow cytometry. (A) Representative examples of unstimulated cells and proliferating cells are shown in the presence of PHA (3 μg/mL) and after coculture with DCs generated from HCs and two AML patients (AML #5, AML #12). (B) Stimulation index of DCs generated from HCs and AML patients with cocktail C, CP, R, and RP (n = 3-9) are summarized as mean ± SD.

Techniques Used: Activation Assay, Staining, Flow Cytometry, Cytometry, Generated

Activation of NK cells by DCs . Allogeneic and autologous non-adherent PBMCs were cocultured with DCs at a ratio of 10:1 for 24 h. Cells were stained for CD3, CD56, CD69 and IFN-γ expression and analyzed by flow cytometry. (A) Shown is the percentage of CD69 expression on CD3 - /CD56 + gated NK cells. Representative histograms of unstimulated NK cells, IL-2 (500 IU/mL) activated NK cells, DCs + autologous NK cells (upper panel) and DCs + allogeneic NK cells (lower panel) of one AML patient are shown (cocktail R was used for DC maturation). The dot blots below show the corresponding intracellular IFN-γ staining, with PMA/ionomycin serving as a positive control. (B) Summary of the percentage of CD69 expression on CD3 - /CD56 + NK cells after coculture with allogeneic and autologous NK cells from HC and AML patients. (C) Production of IFN-γ after coculture of DCs and isolated NK cells was assessed by ELISA. DCs were generated and cocultured with allogeneic NK cells at a ratio of 1:10 for 24 h. Supernatant was analyzed by standard ELISA for IFN-γ. The comparison of HC and AML patients is shown using the four different maturation cocktails. As a positive control NK cells were stimulated with IL-2.
Figure Legend Snippet: Activation of NK cells by DCs . Allogeneic and autologous non-adherent PBMCs were cocultured with DCs at a ratio of 10:1 for 24 h. Cells were stained for CD3, CD56, CD69 and IFN-γ expression and analyzed by flow cytometry. (A) Shown is the percentage of CD69 expression on CD3 - /CD56 + gated NK cells. Representative histograms of unstimulated NK cells, IL-2 (500 IU/mL) activated NK cells, DCs + autologous NK cells (upper panel) and DCs + allogeneic NK cells (lower panel) of one AML patient are shown (cocktail R was used for DC maturation). The dot blots below show the corresponding intracellular IFN-γ staining, with PMA/ionomycin serving as a positive control. (B) Summary of the percentage of CD69 expression on CD3 - /CD56 + NK cells after coculture with allogeneic and autologous NK cells from HC and AML patients. (C) Production of IFN-γ after coculture of DCs and isolated NK cells was assessed by ELISA. DCs were generated and cocultured with allogeneic NK cells at a ratio of 1:10 for 24 h. Supernatant was analyzed by standard ELISA for IFN-γ. The comparison of HC and AML patients is shown using the four different maturation cocktails. As a positive control NK cells were stimulated with IL-2.

Techniques Used: Activation Assay, Staining, Expressing, Flow Cytometry, Cytometry, Positive Control, Isolation, Enzyme-linked Immunosorbent Assay, Generated

3) Product Images from "Age-related differences in humoral and cellular immune responses after primary immunisation: indications for stratified vaccination schedules"

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

Journal: Scientific Reports

doi: 10.1038/s41598-018-28111-8

Vaccine-specific cytokine production. ( a ) IL-2, ( b ) IFN-γ and ( c ) IL-10 production of PBMC after 48 hour stimulation in vitro with JE virus antigen and TBE virus antigen (TBEV), respectively, or incubation with just medium. Cytokine levels were measured in supernatants. ( d ) JE-specific IFN-γ/IL-10 ratio after subtraction of medium values. Statistical analysis by General Linear Model with log-transformed values. Individual comparisons by linear contrasts. * p
Figure Legend Snippet: Vaccine-specific cytokine production. ( a ) IL-2, ( b ) IFN-γ and ( c ) IL-10 production of PBMC after 48 hour stimulation in vitro with JE virus antigen and TBE virus antigen (TBEV), respectively, or incubation with just medium. Cytokine levels were measured in supernatants. ( d ) JE-specific IFN-γ/IL-10 ratio after subtraction of medium values. Statistical analysis by General Linear Model with log-transformed values. Individual comparisons by linear contrasts. * p

Techniques Used: In Vitro, Incubation, Transformation Assay

4) Product Images from "Natalizumab restores aberrant miRNA expression profile in multiple sclerosis and reveals a critical role for miR-20b"

Article Title: Natalizumab restores aberrant miRNA expression profile in multiple sclerosis and reveals a critical role for miR-20b

Journal: Annals of Clinical and Translational Neurology

doi: 10.1002/acn3.152

miR-20b expression in T cells and in PBMC from natalizumab-associated progressive multifocal leukoencephalopathy (PML). (A) PBMC were isolated using a Ficoll gradient and CD4+ T cells were sorted with magnetic beads ( n = 9; randomly selected patients of our initial longitudinal cohort consisting of 17 patients). miR-20b expression (qPCR) was measured in the CD4+ and CD4-negative fraction and compared using t- test. (B) CD4+ cells were activated by plate-bound anti-CD3-antibody and soluble anti-CD28-antibody without further cytokines, termed “Th0”, and with IL-1 (10 ng/mL), IL-6 (10 ng/mL), IL-23 (10 ng/mL) and TGF- β (5 ng/mL), termed “Th17”, as well as freshly isolated CD4+ T cells, termed “fresh”. miR-20b levels were measured by qPCR. (C) miR-20b levels in PBMC from natalizumab-treated patients ( n = 9, left bar) were compared to miR-20b levels in two PBMC samples from patients with natalizumab-associated PML (each patient one bar).
Figure Legend Snippet: miR-20b expression in T cells and in PBMC from natalizumab-associated progressive multifocal leukoencephalopathy (PML). (A) PBMC were isolated using a Ficoll gradient and CD4+ T cells were sorted with magnetic beads ( n = 9; randomly selected patients of our initial longitudinal cohort consisting of 17 patients). miR-20b expression (qPCR) was measured in the CD4+ and CD4-negative fraction and compared using t- test. (B) CD4+ cells were activated by plate-bound anti-CD3-antibody and soluble anti-CD28-antibody without further cytokines, termed “Th0”, and with IL-1 (10 ng/mL), IL-6 (10 ng/mL), IL-23 (10 ng/mL) and TGF- β (5 ng/mL), termed “Th17”, as well as freshly isolated CD4+ T cells, termed “fresh”. miR-20b levels were measured by qPCR. (C) miR-20b levels in PBMC from natalizumab-treated patients ( n = 9, left bar) were compared to miR-20b levels in two PBMC samples from patients with natalizumab-associated PML (each patient one bar).

Techniques Used: Expressing, Isolation, Magnetic Beads, Real-time Polymerase Chain Reaction

5) Product Images from "Antigen delivery to dendritic cells shapes human CD4+ and CD8+ T cell memory responses to Staphylococcus aureus"

Article Title: Antigen delivery to dendritic cells shapes human CD4+ and CD8+ T cell memory responses to Staphylococcus aureus

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006387

Protein-derived antigens activate memory T cells. Human (a) CD4 + or (b) CD8 + T cells were isolated from frozen PBMC via magnetic beads. Cell fractions were either purified for (a) CD14 - CD8 - and (b) CD14 - CD8 + , and CD45RO - CD45RA + (naïve) or CD45RO + CD45RA - (memory) phenotype. Cytokine secretion profiles after 5 days of MoDC/T cell co-culture stimulated with SpA protein were measured by multiplex cytokine array, done in duplicates. TNF, IFNγ, IL-5 and IL-13 are presented as mean ± SEM of n = 7 or 8 donors, respectively. p value refers to the same condition in naïve T cells. p**
Figure Legend Snippet: Protein-derived antigens activate memory T cells. Human (a) CD4 + or (b) CD8 + T cells were isolated from frozen PBMC via magnetic beads. Cell fractions were either purified for (a) CD14 - CD8 - and (b) CD14 - CD8 + , and CD45RO - CD45RA + (naïve) or CD45RO + CD45RA - (memory) phenotype. Cytokine secretion profiles after 5 days of MoDC/T cell co-culture stimulated with SpA protein were measured by multiplex cytokine array, done in duplicates. TNF, IFNγ, IL-5 and IL-13 are presented as mean ± SEM of n = 7 or 8 donors, respectively. p value refers to the same condition in naïve T cells. p**

Techniques Used: Derivative Assay, Isolation, Magnetic Beads, Purification, Co-Culture Assay, Multiplex Assay

mRNA-promoted cytokine response and adjuvant effect. (a) PBMC were co-cultured with autologous MoDC loaded with either non-coding control mRNA (NC), spa mRNA, SpA protein or the lipofection control (LF). IFNγ was detected after overnight incubation by ELISpot. The individual results of the single donors (n = 7) were connected with lines and displayed as IFNγ spots. Analysis was carried out in technical duplicates. Following testing for normal distribution, paired Student’s t-test was used to calculate significance. (b) Cytokine production by MoDC stimulated with mRNA-encoded staphylococcal antigens or the respective proteins and controls (lipofectamine (LF), non-coding control mRNA (NC), Influenza MP1 peptides (MP1) and tetanus toxoid (TT)) was measured in supernatants after 24 hours. The results of duplicates for TNF and IFNα are shown as mean values ± SEM of n = 4 donors. p≥ 0.05 n.s. (One-way ANOVA) (c) Stimulation of MoDC/CD8 + T cell co-cultures with PBP2a or SpA in the presence and absence of NC mRNA after overnight IFNγ ELISPOT analysis. The ELISPOT enzymatic activity relative to the unstimulated control is shown as mean values ± SEM of n = 8 independent donors. Statistical analysis was done using the Wilcoxon matched-pairs signed rank test. Experiments were carried out in duplicates. (d) Upon transfection of MoDC with spa mRNA and co-culture with CD8 + T cells, blocking antibodies against IFNα and TNFα alone or in combination were added and IFNγ secretion of duplicates was quantified by ELISPOT. IFNγ enzymatic activity of n = 5 independent donors is normalized to the unstimulated isotype control and displayed as mean ± SEM. Following testing for normal distribution, paired Student’s t-test was used to calculate significance. p**
Figure Legend Snippet: mRNA-promoted cytokine response and adjuvant effect. (a) PBMC were co-cultured with autologous MoDC loaded with either non-coding control mRNA (NC), spa mRNA, SpA protein or the lipofection control (LF). IFNγ was detected after overnight incubation by ELISpot. The individual results of the single donors (n = 7) were connected with lines and displayed as IFNγ spots. Analysis was carried out in technical duplicates. Following testing for normal distribution, paired Student’s t-test was used to calculate significance. (b) Cytokine production by MoDC stimulated with mRNA-encoded staphylococcal antigens or the respective proteins and controls (lipofectamine (LF), non-coding control mRNA (NC), Influenza MP1 peptides (MP1) and tetanus toxoid (TT)) was measured in supernatants after 24 hours. The results of duplicates for TNF and IFNα are shown as mean values ± SEM of n = 4 donors. p≥ 0.05 n.s. (One-way ANOVA) (c) Stimulation of MoDC/CD8 + T cell co-cultures with PBP2a or SpA in the presence and absence of NC mRNA after overnight IFNγ ELISPOT analysis. The ELISPOT enzymatic activity relative to the unstimulated control is shown as mean values ± SEM of n = 8 independent donors. Statistical analysis was done using the Wilcoxon matched-pairs signed rank test. Experiments were carried out in duplicates. (d) Upon transfection of MoDC with spa mRNA and co-culture with CD8 + T cells, blocking antibodies against IFNα and TNFα alone or in combination were added and IFNγ secretion of duplicates was quantified by ELISPOT. IFNγ enzymatic activity of n = 5 independent donors is normalized to the unstimulated isotype control and displayed as mean ± SEM. Following testing for normal distribution, paired Student’s t-test was used to calculate significance. p**

Techniques Used: Cell Culture, Incubation, Enzyme-linked Immunospot, Activity Assay, Transfection, Co-Culture Assay, Blocking Assay

6) Product Images from "Impaired miRNA degradation by post-transcriptional addition of 3’ cytosine and adenine in T cell activation"

Article Title: Impaired miRNA degradation by post-transcriptional addition of 3’ cytosine and adenine in T cell activation

Journal: bioRxiv

doi: 10.1101/2020.08.19.257816

Differential miRNA expression 3h, 6h and 24h after IFN I stimulation of human primary CD4+ T cells. A) Heat map for differentially expressed miRNAs. The heatmap represents relative expression values for a non-redundant collection of differentially expressed miRNAs (adjusted p-value
Figure Legend Snippet: Differential miRNA expression 3h, 6h and 24h after IFN I stimulation of human primary CD4+ T cells. A) Heat map for differentially expressed miRNAs. The heatmap represents relative expression values for a non-redundant collection of differentially expressed miRNAs (adjusted p-value

Techniques Used: Expressing

Differential miRNA expression 3h, 6h and 24h after αCD3αCD28 stimulation of human primary CD4+ T cells. A) Heat map for differentially expressed miRNAs. The heatmap represents relative expression values for a non-redundant collection of differentially expressed miRNAs (adjusted p-value
Figure Legend Snippet: Differential miRNA expression 3h, 6h and 24h after αCD3αCD28 stimulation of human primary CD4+ T cells. A) Heat map for differentially expressed miRNAs. The heatmap represents relative expression values for a non-redundant collection of differentially expressed miRNAs (adjusted p-value

Techniques Used: Expressing

Expression of uridylated RNA degrading enzymes (Dis3L2 and Eri1) and terminal uridyl transferases (TUT4 y TUT7) upon CD4+ T cell activation. Western blot analysis of protein expression in human primary CD4+ T cell stimulated with αCD3αCD28, assessing Dis3L2 (A), Eri1 (B), TUT4 (C) and TUT7 (D). Fold increase compared to non-stimulation was represented for each donor to observe evolution upon activation (left panel) and using group median and interquartile range with whiskers ranging from minimum to maximum values (middle panel). Right panels include two examples for each protein to highlight inter-donor variability in upregulation kinetics. Band intensities were normalized to ERMs values and relativized to unstimulated conditions. Statistical analysis: Kruskal-Wallis test, Dunn’s multiple comparisons test [* p-value
Figure Legend Snippet: Expression of uridylated RNA degrading enzymes (Dis3L2 and Eri1) and terminal uridyl transferases (TUT4 y TUT7) upon CD4+ T cell activation. Western blot analysis of protein expression in human primary CD4+ T cell stimulated with αCD3αCD28, assessing Dis3L2 (A), Eri1 (B), TUT4 (C) and TUT7 (D). Fold increase compared to non-stimulation was represented for each donor to observe evolution upon activation (left panel) and using group median and interquartile range with whiskers ranging from minimum to maximum values (middle panel). Right panels include two examples for each protein to highlight inter-donor variability in upregulation kinetics. Band intensities were normalized to ERMs values and relativized to unstimulated conditions. Statistical analysis: Kruskal-Wallis test, Dunn’s multiple comparisons test [* p-value

Techniques Used: Expressing, Activation Assay, Western Blot

7) Product Images from "New insights into tenocyte-immune cell interplay in an in vitro model of inflammation"

Article Title: New insights into tenocyte-immune cell interplay in an in vitro model of inflammation

Journal: Scientific Reports

doi: 10.1038/s41598-017-09875-x

Tenocytes produce IL-6 upon stimulation. Cytokines were measured in media from αCD3αCD28 stimulated PBMCs (stim. media) (see Fig. 1a ) using the Legendplex TM human inflammation panel. After tenocytes were cultured for 3 days with either the control media from unstimulated PBMC (unstim. media + tenocytes; dotted line) or stimulation media (stim. media + tenocytes), cytokines were measured in the supernatants. The levels of IL-6 ( a ), IL-8 ( b ), MCP-1 ( c ) and IFNγ ( d ) are shown for the tenocytes cultured with stimulated media compared to the stimulated media without tenocytes present. The median levels for tenocytes incubated with the unstimulated media are shown next to the dotted line at that level on a–c , and IFNγ was undetectable. Samples were treated following the manufacturer’s instructions and measured by flow cytometry. Analysis was done using Legendplex software. Data are presented as the median with the interquartile range with an n = 5 for stimulated media alone and n = 10 for stimulated media + tenocytes, and are considered significantly different when *p
Figure Legend Snippet: Tenocytes produce IL-6 upon stimulation. Cytokines were measured in media from αCD3αCD28 stimulated PBMCs (stim. media) (see Fig. 1a ) using the Legendplex TM human inflammation panel. After tenocytes were cultured for 3 days with either the control media from unstimulated PBMC (unstim. media + tenocytes; dotted line) or stimulation media (stim. media + tenocytes), cytokines were measured in the supernatants. The levels of IL-6 ( a ), IL-8 ( b ), MCP-1 ( c ) and IFNγ ( d ) are shown for the tenocytes cultured with stimulated media compared to the stimulated media without tenocytes present. The median levels for tenocytes incubated with the unstimulated media are shown next to the dotted line at that level on a–c , and IFNγ was undetectable. Samples were treated following the manufacturer’s instructions and measured by flow cytometry. Analysis was done using Legendplex software. Data are presented as the median with the interquartile range with an n = 5 for stimulated media alone and n = 10 for stimulated media + tenocytes, and are considered significantly different when *p

Techniques Used: Cell Culture, Incubation, Flow Cytometry, Cytometry, Software

Potential role of cross-talk between tenocytes and macrophages in an inflammatory milieu in tendinopathy. The scheme illustrates the effects of inflammatory conditions on human tenocytes alone and during their interplay with human macrophages on surface marker expression and cytokine release patterns. A mixed cytokine stimulation originating from αCD3αCD28 activated human PBMCs including all subsets (T cells, B cells, NK cells and monocytes (Mo)) induces the up-regulation of HLA-molecules (HLA-DR, HLA-ABC) and adhesion molecules (ICAM-1, VCAM-1) on tenocytes and enhances their IL-6 secretion. After co-culturing monocyte-derived macrophages (Mϕ) with pre-stimulated tenocytes, the macrophages display a mixed M1/M2 macrophage phenotype with enhanced expression of CD80 (characteristic for M1-Mϕ), but reduced HLA-DR (characteristic for M2-Mϕ). As yet unknown soluble factors lead to higher levels of IL-6, IL-8 and MCP-1 secretion. We hypothesize, that this interaction might contribute to a disturbed resolution of the immune response after tendon injury leading to chronic tendinopathies.
Figure Legend Snippet: Potential role of cross-talk between tenocytes and macrophages in an inflammatory milieu in tendinopathy. The scheme illustrates the effects of inflammatory conditions on human tenocytes alone and during their interplay with human macrophages on surface marker expression and cytokine release patterns. A mixed cytokine stimulation originating from αCD3αCD28 activated human PBMCs including all subsets (T cells, B cells, NK cells and monocytes (Mo)) induces the up-regulation of HLA-molecules (HLA-DR, HLA-ABC) and adhesion molecules (ICAM-1, VCAM-1) on tenocytes and enhances their IL-6 secretion. After co-culturing monocyte-derived macrophages (Mϕ) with pre-stimulated tenocytes, the macrophages display a mixed M1/M2 macrophage phenotype with enhanced expression of CD80 (characteristic for M1-Mϕ), but reduced HLA-DR (characteristic for M2-Mϕ). As yet unknown soluble factors lead to higher levels of IL-6, IL-8 and MCP-1 secretion. We hypothesize, that this interaction might contribute to a disturbed resolution of the immune response after tendon injury leading to chronic tendinopathies.

Techniques Used: Marker, Expressing, Derivative Assay

Pro-inflammatory stimulation media increases the size and granularity of tenocytes, and changes their surface marker expression. Following incubation for 3 days with either the control media from unstimulated PBMC (unstim. media) or the pro-inflammatory stimulation media from αCD3αCD28 stimulated PBMCs (stim. media) ( a ), supernatants were collected for later cytokine evaluation before the tenocytes were stained with a panel of human-specific antibodies to characteristic surface markers and analyzed by flow cytometry. Tenocytes included in the viable gate with unstim. media ( b ) or with the stim. media ( c ) are shown here with representative flow cytometry density plots. The mean fluorescence intensity (MFI) of forward scatter area (FSC-A) and sidewards scatter area (SSC-A) indicating cell size and granularity are shown ( d ). The expression of the adhesion molecules ICAM-1 (CD54) and VCAM-1 (CD106), as well as the HLA class I molecule HLA-ABC and the HLA class II molecule HLA-DR ( e ) and CD90 (Thy-1) ( f ) are also shown after incubation with unstimulated and stimulated media. Data are presented as the median of n = 10 with the interquartile range and are considered significantly different when *p
Figure Legend Snippet: Pro-inflammatory stimulation media increases the size and granularity of tenocytes, and changes their surface marker expression. Following incubation for 3 days with either the control media from unstimulated PBMC (unstim. media) or the pro-inflammatory stimulation media from αCD3αCD28 stimulated PBMCs (stim. media) ( a ), supernatants were collected for later cytokine evaluation before the tenocytes were stained with a panel of human-specific antibodies to characteristic surface markers and analyzed by flow cytometry. Tenocytes included in the viable gate with unstim. media ( b ) or with the stim. media ( c ) are shown here with representative flow cytometry density plots. The mean fluorescence intensity (MFI) of forward scatter area (FSC-A) and sidewards scatter area (SSC-A) indicating cell size and granularity are shown ( d ). The expression of the adhesion molecules ICAM-1 (CD54) and VCAM-1 (CD106), as well as the HLA class I molecule HLA-ABC and the HLA class II molecule HLA-DR ( e ) and CD90 (Thy-1) ( f ) are also shown after incubation with unstimulated and stimulated media. Data are presented as the median of n = 10 with the interquartile range and are considered significantly different when *p

Techniques Used: Marker, Expressing, Incubation, Staining, Flow Cytometry, Cytometry, Fluorescence

8) Product Images from "Effects of TLR agonists on maturation and function of 3-day dendritic cells from AML patients in complete remission"

Article Title: Effects of TLR agonists on maturation and function of 3-day dendritic cells from AML patients in complete remission

Journal: Journal of Translational Medicine

doi: 10.1186/1479-5876-9-151

Activation of allogeneic T cells by DCs . PBMCs from healthy unrelated donors were stained with CFSE and cocultured with DCs at a ratio of 10:1. After 6 days, proliferation was assessed by flow cytometry. (A) Representative examples of unstimulated cells and proliferating cells are shown in the presence of PHA (3 μg/mL) and after coculture with DCs generated from HCs and two AML patients (AML #5, AML #12). (B) Stimulation index of DCs generated from HCs and AML patients with cocktail C, CP, R, and RP (n = 3-9) are summarized as mean ± SD.
Figure Legend Snippet: Activation of allogeneic T cells by DCs . PBMCs from healthy unrelated donors were stained with CFSE and cocultured with DCs at a ratio of 10:1. After 6 days, proliferation was assessed by flow cytometry. (A) Representative examples of unstimulated cells and proliferating cells are shown in the presence of PHA (3 μg/mL) and after coculture with DCs generated from HCs and two AML patients (AML #5, AML #12). (B) Stimulation index of DCs generated from HCs and AML patients with cocktail C, CP, R, and RP (n = 3-9) are summarized as mean ± SD.

Techniques Used: Activation Assay, Staining, Flow Cytometry, Cytometry, Generated

Activation of NK cells by DCs . Allogeneic and autologous non-adherent PBMCs were cocultured with DCs at a ratio of 10:1 for 24 h. Cells were stained for CD3, CD56, CD69 and IFN-γ expression and analyzed by flow cytometry. (A) Shown is the percentage of CD69 expression on CD3 - /CD56 + gated NK cells. Representative histograms of unstimulated NK cells, IL-2 (500 IU/mL) activated NK cells, DCs + autologous NK cells (upper panel) and DCs + allogeneic NK cells (lower panel) of one AML patient are shown (cocktail R was used for DC maturation). The dot blots below show the corresponding intracellular IFN-γ staining, with PMA/ionomycin serving as a positive control. (B) Summary of the percentage of CD69 expression on CD3 - /CD56 + NK cells after coculture with allogeneic and autologous NK cells from HC and AML patients. (C) Production of IFN-γ after coculture of DCs and isolated NK cells was assessed by ELISA. DCs were generated and cocultured with allogeneic NK cells at a ratio of 1:10 for 24 h. Supernatant was analyzed by standard ELISA for IFN-γ. The comparison of HC and AML patients is shown using the four different maturation cocktails. As a positive control NK cells were stimulated with IL-2.
Figure Legend Snippet: Activation of NK cells by DCs . Allogeneic and autologous non-adherent PBMCs were cocultured with DCs at a ratio of 10:1 for 24 h. Cells were stained for CD3, CD56, CD69 and IFN-γ expression and analyzed by flow cytometry. (A) Shown is the percentage of CD69 expression on CD3 - /CD56 + gated NK cells. Representative histograms of unstimulated NK cells, IL-2 (500 IU/mL) activated NK cells, DCs + autologous NK cells (upper panel) and DCs + allogeneic NK cells (lower panel) of one AML patient are shown (cocktail R was used for DC maturation). The dot blots below show the corresponding intracellular IFN-γ staining, with PMA/ionomycin serving as a positive control. (B) Summary of the percentage of CD69 expression on CD3 - /CD56 + NK cells after coculture with allogeneic and autologous NK cells from HC and AML patients. (C) Production of IFN-γ after coculture of DCs and isolated NK cells was assessed by ELISA. DCs were generated and cocultured with allogeneic NK cells at a ratio of 1:10 for 24 h. Supernatant was analyzed by standard ELISA for IFN-γ. The comparison of HC and AML patients is shown using the four different maturation cocktails. As a positive control NK cells were stimulated with IL-2.

Techniques Used: Activation Assay, Staining, Expressing, Flow Cytometry, Cytometry, Positive Control, Isolation, Enzyme-linked Immunosorbent Assay, Generated

9) Product Images from "CD96 expression determines the inflammatory potential of IL-9–producing Th9 cells"

Article Title: CD96 expression determines the inflammatory potential of IL-9–producing Th9 cells

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

doi: 10.1073/pnas.1708329115

In vitro differentiated Th9 cells display a unique activation phenotype with heterogeneous cytokine and gene expression. ( A ) Nonsupervised hierarchical clustering analysis of differentially expressed genes encoding for cell surface expressed molecules in nonpolarized (w/o), Th1, or Th9 polarized alloreactive C57BL/6 T cells. Gene expression of three biological replicates (enriched for CD44 + cells) was determined by PCR array and normalized to B2m expression. White areas indicate undetermined (Ct > 35) values, and genes written in red were stained for protein expression as shown in B . ( B ) Histograms comparing protein expression of surface molecules associated with T-cell activation in nonactivated (CD44 − ) and activated (CD44 + ) alloreactive BALB/c T cells from PMA/ionomycin-stimulated cocultures. ( C ) Staining of intracellular cytokines in alloreactive BALB/c CD44 + Th9 cells. The P value of nine independent experiments was determined by one-tailed Wilcoxon matched-pairs signed rank test. ( D ) Nonsupervised hierarchical clustering analysis of differentially expressed genes encoding for cell surface relevant molecules in fluorescence activated cell (FAC)-sorted IL-9 + , IL-4 + , and double-negative (DN) cells. Cells were sorted from PMA/ionomycin-restimulated alloreactive Th9 cocultures with total BALB/c CD4 + T cells. The results of three biological replicates are shown (IL-4 + samples had to be pooled due to limited cell number).
Figure Legend Snippet: In vitro differentiated Th9 cells display a unique activation phenotype with heterogeneous cytokine and gene expression. ( A ) Nonsupervised hierarchical clustering analysis of differentially expressed genes encoding for cell surface expressed molecules in nonpolarized (w/o), Th1, or Th9 polarized alloreactive C57BL/6 T cells. Gene expression of three biological replicates (enriched for CD44 + cells) was determined by PCR array and normalized to B2m expression. White areas indicate undetermined (Ct > 35) values, and genes written in red were stained for protein expression as shown in B . ( B ) Histograms comparing protein expression of surface molecules associated with T-cell activation in nonactivated (CD44 − ) and activated (CD44 + ) alloreactive BALB/c T cells from PMA/ionomycin-stimulated cocultures. ( C ) Staining of intracellular cytokines in alloreactive BALB/c CD44 + Th9 cells. The P value of nine independent experiments was determined by one-tailed Wilcoxon matched-pairs signed rank test. ( D ) Nonsupervised hierarchical clustering analysis of differentially expressed genes encoding for cell surface relevant molecules in fluorescence activated cell (FAC)-sorted IL-9 + , IL-4 + , and double-negative (DN) cells. Cells were sorted from PMA/ionomycin-restimulated alloreactive Th9 cocultures with total BALB/c CD4 + T cells. The results of three biological replicates are shown (IL-4 + samples had to be pooled due to limited cell number).

Techniques Used: In Vitro, Activation Assay, Expressing, Polymerase Chain Reaction, Staining, One-tailed Test, Fluorescence

Transfer of Th9 cells into B cell- and T cell-deficient Rag1 −/− mice causes graft rejection, weight loss, and intestinal inflammation. ( A – D ) C57BL/6 Rag1 −/− mice received 1 or 2 × 10 5 activated (CD44 + ) T cells sorted from in vitro differentiated alloreactive C57BL/6 Th1 ( n = 4–5) or Th9 ( n = 4–7) cultures and a BALB/c skin graft on the following day. ( A ) Survival of BALB/c donor skin. A Log-rank (Mantel–Cox) test was applied. ( B ) Body weight of mice depicted as mean ± SEM. P value of the interaction term (group with time) was calculated using an ANOVA type III test after fitting a linear mixed-effect model to the body weight data. ( C ) Representative hematoxylin and eosin stained ileum sections collected on day 12 (w/o) or 13 (Th1, Th9) post skin transplantation. (Scale bar: 100 µm.) ( D ) Villus height-to-crypt depth ratio in ileal mucosa with median value. For statistical analysis, a one-tailed Mann–Whitney test was applied. ( E ) Staining of in vitro differentiated alloreactive (CD44 + ) BALB/c T cells for intracellular expression of signature cytokines. Cells were cultured for 3 d in the absence (w/o) or presence of Th9, Th1, or Th2 polarizing cytokines. ( F ) Intracellular staining for IL-9 in activated (1st activ.) and (with allogeneic dendritic cells) reactivated (2nd activ.) in vitro differentiated alloreactive C57BL/6 Th9 cells ( n = 4). Statistical analysis by Kruskal–Wallis test. n.s., not significant ( P > 0.05).
Figure Legend Snippet: Transfer of Th9 cells into B cell- and T cell-deficient Rag1 −/− mice causes graft rejection, weight loss, and intestinal inflammation. ( A – D ) C57BL/6 Rag1 −/− mice received 1 or 2 × 10 5 activated (CD44 + ) T cells sorted from in vitro differentiated alloreactive C57BL/6 Th1 ( n = 4–5) or Th9 ( n = 4–7) cultures and a BALB/c skin graft on the following day. ( A ) Survival of BALB/c donor skin. A Log-rank (Mantel–Cox) test was applied. ( B ) Body weight of mice depicted as mean ± SEM. P value of the interaction term (group with time) was calculated using an ANOVA type III test after fitting a linear mixed-effect model to the body weight data. ( C ) Representative hematoxylin and eosin stained ileum sections collected on day 12 (w/o) or 13 (Th1, Th9) post skin transplantation. (Scale bar: 100 µm.) ( D ) Villus height-to-crypt depth ratio in ileal mucosa with median value. For statistical analysis, a one-tailed Mann–Whitney test was applied. ( E ) Staining of in vitro differentiated alloreactive (CD44 + ) BALB/c T cells for intracellular expression of signature cytokines. Cells were cultured for 3 d in the absence (w/o) or presence of Th9, Th1, or Th2 polarizing cytokines. ( F ) Intracellular staining for IL-9 in activated (1st activ.) and (with allogeneic dendritic cells) reactivated (2nd activ.) in vitro differentiated alloreactive C57BL/6 Th9 cells ( n = 4). Statistical analysis by Kruskal–Wallis test. n.s., not significant ( P > 0.05).

Techniques Used: Mouse Assay, In Vitro, Staining, Transplantation Assay, One-tailed Test, MANN-WHITNEY, Expressing, Cell Culture

10) Product Images from "CD96 expression determines the inflammatory potential of IL-9–producing Th9 cells"

Article Title: CD96 expression determines the inflammatory potential of IL-9–producing Th9 cells

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

doi: 10.1073/pnas.1708329115

In vitro differentiated Th9 cells display a unique activation phenotype with heterogeneous cytokine and gene expression. ( A ) Nonsupervised hierarchical clustering analysis of differentially expressed genes encoding for cell surface expressed molecules in nonpolarized (w/o), Th1, or Th9 polarized alloreactive C57BL/6 T cells. Gene expression of three biological replicates (enriched for CD44 +  cells) was determined by PCR array and normalized to  B2m  expression. White areas indicate undetermined (Ct  >  35) values, and genes written in red were stained for protein expression as shown in  B . ( B ) Histograms comparing protein expression of surface molecules associated with T-cell activation in nonactivated (CD44 − ) and activated (CD44 + ) alloreactive BALB/c T cells from PMA/ionomycin-stimulated cocultures. ( C ) Staining of intracellular cytokines in alloreactive BALB/c CD44 +  Th9 cells. The  P  value of nine independent experiments was determined by one-tailed Wilcoxon matched-pairs signed rank test. ( D ) Nonsupervised hierarchical clustering analysis of differentially expressed genes encoding for cell surface relevant molecules in fluorescence activated cell (FAC)-sorted IL-9 + , IL-4 + , and double-negative (DN) cells. Cells were sorted from PMA/ionomycin-restimulated alloreactive Th9 cocultures with total BALB/c CD4 +  T cells. The results of three biological replicates are shown (IL-4 +  samples had to be pooled due to limited cell number).
Figure Legend Snippet: In vitro differentiated Th9 cells display a unique activation phenotype with heterogeneous cytokine and gene expression. ( A ) Nonsupervised hierarchical clustering analysis of differentially expressed genes encoding for cell surface expressed molecules in nonpolarized (w/o), Th1, or Th9 polarized alloreactive C57BL/6 T cells. Gene expression of three biological replicates (enriched for CD44 + cells) was determined by PCR array and normalized to B2m expression. White areas indicate undetermined (Ct > 35) values, and genes written in red were stained for protein expression as shown in B . ( B ) Histograms comparing protein expression of surface molecules associated with T-cell activation in nonactivated (CD44 − ) and activated (CD44 + ) alloreactive BALB/c T cells from PMA/ionomycin-stimulated cocultures. ( C ) Staining of intracellular cytokines in alloreactive BALB/c CD44 + Th9 cells. The P value of nine independent experiments was determined by one-tailed Wilcoxon matched-pairs signed rank test. ( D ) Nonsupervised hierarchical clustering analysis of differentially expressed genes encoding for cell surface relevant molecules in fluorescence activated cell (FAC)-sorted IL-9 + , IL-4 + , and double-negative (DN) cells. Cells were sorted from PMA/ionomycin-restimulated alloreactive Th9 cocultures with total BALB/c CD4 + T cells. The results of three biological replicates are shown (IL-4 + samples had to be pooled due to limited cell number).

Techniques Used: In Vitro, Activation Assay, Expressing, Polymerase Chain Reaction, Staining, One-tailed Test, Fluorescence

Single cell profiling identifies CD96 high  and CD96 low  subsets of Th9 cells. ( A – D ) Single cell gene expression analysis of CD44 +  alloreactive T cells sorted from nonpolarized (w/o;  n  = 100), Th1 ( n  = 67), and Th9 ( n  = 113) polarized BALB/c cocultures. See  Table S2  for a list of all genes measured. ( A ) Principal component analysis of gene expression in indicated Th subsets. ( B ) Nonsupervised hierarchical clustering analysis (Pearson’s correlation) comparing gene expression in Th subsets. Analysis includes all genes with detectable expression. ( C ) Box plots of most differentially expressed genes. Bonferroni-corrected ANOVA  P  values:  Il9  ( P  = 1.4 × 10 −126 ),  Il2ra  ( P  = 1.4 × 10 −65 ),  Cd83  ( P  = 7.1 × 10 −64 ),  Il4ra  ( P  = 1.2 × 10 −52 ),  Gata3  ( P  = 3.6 × 10 −47 ),  Il1r1  ( P  = 5.5 × 10 −47 ),  Ccr4  ( P  = 2.2 × 10 −46 ), and  Cd28  ( P  = 2.3 × 10 −43 ). ( D ) Nonsupervised hierarchical clustering analysis (complete linkage method) of gene expression in CD44 +  T cells sorted from alloreactive BALB/c Th9 cultures. Displayed genes contributed to clustering. Frame indicates a cluster of Th9 cells characterized by high  Cd96  expression. ( E ) Contour plots of surface CD96 expression in CD44 +  polarized alloreactive BALB/c T cells, including fluorescence minus one (FMO) staining control (Ctrl). ( F ) Comparison of CD96 expression levels in IL-9 +  and IFN-γ +  human CD4 +  T cells. PBMCs from blood of 10 healthy individuals were stimulated ex vivo with  Staphylococcus  enterotoxin B (SEB) for 24 h. A one-tailed Wilcoxon matched-pairs signed rank test was applied. gMFI, geometric mean fluorescence intensity.
Figure Legend Snippet: Single cell profiling identifies CD96 high and CD96 low subsets of Th9 cells. ( A – D ) Single cell gene expression analysis of CD44 + alloreactive T cells sorted from nonpolarized (w/o; n = 100), Th1 ( n = 67), and Th9 ( n = 113) polarized BALB/c cocultures. See Table S2 for a list of all genes measured. ( A ) Principal component analysis of gene expression in indicated Th subsets. ( B ) Nonsupervised hierarchical clustering analysis (Pearson’s correlation) comparing gene expression in Th subsets. Analysis includes all genes with detectable expression. ( C ) Box plots of most differentially expressed genes. Bonferroni-corrected ANOVA P values: Il9 ( P = 1.4 × 10 −126 ), Il2ra ( P = 1.4 × 10 −65 ), Cd83 ( P = 7.1 × 10 −64 ), Il4ra ( P = 1.2 × 10 −52 ), Gata3 ( P = 3.6 × 10 −47 ), Il1r1 ( P = 5.5 × 10 −47 ), Ccr4 ( P = 2.2 × 10 −46 ), and Cd28 ( P = 2.3 × 10 −43 ). ( D ) Nonsupervised hierarchical clustering analysis (complete linkage method) of gene expression in CD44 + T cells sorted from alloreactive BALB/c Th9 cultures. Displayed genes contributed to clustering. Frame indicates a cluster of Th9 cells characterized by high Cd96 expression. ( E ) Contour plots of surface CD96 expression in CD44 + polarized alloreactive BALB/c T cells, including fluorescence minus one (FMO) staining control (Ctrl). ( F ) Comparison of CD96 expression levels in IL-9 + and IFN-γ + human CD4 + T cells. PBMCs from blood of 10 healthy individuals were stimulated ex vivo with Staphylococcus enterotoxin B (SEB) for 24 h. A one-tailed Wilcoxon matched-pairs signed rank test was applied. gMFI, geometric mean fluorescence intensity.

Techniques Used: Expressing, Fluorescence, Staining, Ex Vivo, One-tailed Test

11) Product Images from "Obesity and Sex Affect the Immune Responses to Tick-Borne Encephalitis Booster Vaccination"

Article Title: Obesity and Sex Affect the Immune Responses to Tick-Borne Encephalitis Booster Vaccination

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.00860

Quantification of naïve and memory sub-populations of B lymphocytes and plasma blasts. Peripheral blood mononuclear cells were stained with fluorochrome-labeled mAbs and analyzed on a BD FACS Canto II flow cytometer. (A) Scatterplot of CD19 + B cells with expression of IgD and CD27 to distinguish naïve, un-switched, switched and double negative (DN) subsets. (B) Quantification of total CD19 + B-cells and of (B1) naïve, (B2) un-switched, and (B3) switched subset before (d0) and one week (d7) after TBE booster vaccination; line is arithmetic mean. (C) Plasma blasts (CD19 + /CD27 ++ /CD38 high ) as percentages of total CD19 + B cells before (d0) and 1 week (d7) after booster; line is arithmetic mean. ANOVA with linear contrasts; *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.
Figure Legend Snippet: Quantification of naïve and memory sub-populations of B lymphocytes and plasma blasts. Peripheral blood mononuclear cells were stained with fluorochrome-labeled mAbs and analyzed on a BD FACS Canto II flow cytometer. (A) Scatterplot of CD19 + B cells with expression of IgD and CD27 to distinguish naïve, un-switched, switched and double negative (DN) subsets. (B) Quantification of total CD19 + B-cells and of (B1) naïve, (B2) un-switched, and (B3) switched subset before (d0) and one week (d7) after TBE booster vaccination; line is arithmetic mean. (C) Plasma blasts (CD19 + /CD27 ++ /CD38 high ) as percentages of total CD19 + B cells before (d0) and 1 week (d7) after booster; line is arithmetic mean. ANOVA with linear contrasts; *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.

Techniques Used: Staining, Labeling, FACS, Flow Cytometry, Expressing

Quantification of circulating T follicular helper cells (cTfh) and cTfh1, cTfh2, and cTfh17 subsets. Peripheral blood mononuclear cells were stained with fluorochrome-labeled mAbs and analyzed on a BD FACS Canto II flow cytometer. (A) Scatterplot of total cTfh (CD3 + /CD4 + /CD45RA − /CXCR5 + ) with respective expression of CXCR3 and/or CCR6 to distinguish cTfh1, cTfh2, and cTfh17 subsets. CXCR3 + CCR6 − cell are cTfh1 cells; CXCR3 − CCR6 − are cTfh2 cells, and CXCR3 − CCR6 + cells are cTfh17 cells. (B) Quantification of cTfh (CD3 + /CD4 + /CD45RA − /CXCR5+) before (d0) and 1 week (d7) after booster vaccination as % of total CD4 + T cells. (C) cTfh1 and (D) cTfh17 cells as % of total cTfh before (d0) and 1 week (d7) after booster; line is arithmetic mean. (E) cTfh17 cells as % of total cTfh before TBE booster (d0) in females and males. Analysis of variance with linear contrasts; * p ≤ 0.05; *** p ≤ 0.05.
Figure Legend Snippet: Quantification of circulating T follicular helper cells (cTfh) and cTfh1, cTfh2, and cTfh17 subsets. Peripheral blood mononuclear cells were stained with fluorochrome-labeled mAbs and analyzed on a BD FACS Canto II flow cytometer. (A) Scatterplot of total cTfh (CD3 + /CD4 + /CD45RA − /CXCR5 + ) with respective expression of CXCR3 and/or CCR6 to distinguish cTfh1, cTfh2, and cTfh17 subsets. CXCR3 + CCR6 − cell are cTfh1 cells; CXCR3 − CCR6 − are cTfh2 cells, and CXCR3 − CCR6 + cells are cTfh17 cells. (B) Quantification of cTfh (CD3 + /CD4 + /CD45RA − /CXCR5+) before (d0) and 1 week (d7) after booster vaccination as % of total CD4 + T cells. (C) cTfh1 and (D) cTfh17 cells as % of total cTfh before (d0) and 1 week (d7) after booster; line is arithmetic mean. (E) cTfh17 cells as % of total cTfh before TBE booster (d0) in females and males. Analysis of variance with linear contrasts; * p ≤ 0.05; *** p ≤ 0.05.

Techniques Used: Staining, Labeling, FACS, Flow Cytometry, Expressing

Cytokine production of re-stimulated PBMCs. Interleukin 2 and IFN-γ concentrations were measured in PBMC culture supernatants with Luminex technology. Geometric mean (GM) concentrations (pg/mL) with 95% CI from PBMCs obtained before (d0) and 1 week (d7) after booster are shown; cytokine concentrations obtained in media-only cultures were subtracted. (A,B) Interleukin 2 and IFN-γ concentrations (pg/ml) from PBMC incubated with 0.48 μg/mL TBE antigen for 48 h, and (C) IL-2 concentrations (pg/mL) from PBMCs incubated with 1 μg/mL Staphylococcus Enterotoxin B for 48 h. ANOVA with linear contrasts; ** p ≤ 0.01, * p ≤ 0.05.
Figure Legend Snippet: Cytokine production of re-stimulated PBMCs. Interleukin 2 and IFN-γ concentrations were measured in PBMC culture supernatants with Luminex technology. Geometric mean (GM) concentrations (pg/mL) with 95% CI from PBMCs obtained before (d0) and 1 week (d7) after booster are shown; cytokine concentrations obtained in media-only cultures were subtracted. (A,B) Interleukin 2 and IFN-γ concentrations (pg/ml) from PBMC incubated with 0.48 μg/mL TBE antigen for 48 h, and (C) IL-2 concentrations (pg/mL) from PBMCs incubated with 1 μg/mL Staphylococcus Enterotoxin B for 48 h. ANOVA with linear contrasts; ** p ≤ 0.01, * p ≤ 0.05.

Techniques Used: Luminex, Incubation

12) Product Images from "SIVcol Nef counteracts SERINC5 by promoting its proteasomal degradation but does not efficiently enhance HIV-1 replication in human CD4+ T cells and lymphoid tissue"

Article Title: SIVcol Nef counteracts SERINC5 by promoting its proteasomal degradation but does not efficiently enhance HIV-1 replication in human CD4+ T cells and lymphoid tissue

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007269

SERINC5 antagonism does not enhance viral replication in primary human CD4+ T cell cultures. (A) Stimulated primary CD4+ T cells were transduced with equal p24 amounts of VSV-G pseudotyped NL4-3 viruses carrying the indicated HIV/SIV  nef  gene or mutant thereof. Cells were maintained in culture for up to 10 days post-transduction under static conditions (left panel) or with agitation (right panel) to limit cell-to-cell viral spread. Every 2 days, samples of cell culture medium from triplicate wells were harvested to determine infectious virus yield by TZM-bl reporter cell assay and CD4+ T cell infection rates were assessed by intracellular p24 staining followed by flow cytometric analysis. Data shown represents measurements obtained from 3 donors (mean ±SEM) . (B) Correlation between the infection rates of TZM-bl and primary CD4+ T cells infected with HIV-1 IRES-eGFP  nef  recombinants produced in the presence of transiently expressed SERINC5 in HEK293T cells. (C) The infectious virus and p24 antigen yields at 4, 6 and 8 days post-infection in the standard CD4+ T cell infection experiments shown in panel A were determined by TZM-bl infection and p24 antigen ELISA, respectively. Virion infectivity normalized for p24 content is shown relative to the  nef -defective HIV-1 construct (100%). Shown are average values obtained at three time points for the three blood donors (+SEM). *p
Figure Legend Snippet: SERINC5 antagonism does not enhance viral replication in primary human CD4+ T cell cultures. (A) Stimulated primary CD4+ T cells were transduced with equal p24 amounts of VSV-G pseudotyped NL4-3 viruses carrying the indicated HIV/SIV nef gene or mutant thereof. Cells were maintained in culture for up to 10 days post-transduction under static conditions (left panel) or with agitation (right panel) to limit cell-to-cell viral spread. Every 2 days, samples of cell culture medium from triplicate wells were harvested to determine infectious virus yield by TZM-bl reporter cell assay and CD4+ T cell infection rates were assessed by intracellular p24 staining followed by flow cytometric analysis. Data shown represents measurements obtained from 3 donors (mean ±SEM) . (B) Correlation between the infection rates of TZM-bl and primary CD4+ T cells infected with HIV-1 IRES-eGFP nef recombinants produced in the presence of transiently expressed SERINC5 in HEK293T cells. (C) The infectious virus and p24 antigen yields at 4, 6 and 8 days post-infection in the standard CD4+ T cell infection experiments shown in panel A were determined by TZM-bl infection and p24 antigen ELISA, respectively. Virion infectivity normalized for p24 content is shown relative to the nef -defective HIV-1 construct (100%). Shown are average values obtained at three time points for the three blood donors (+SEM). *p

Techniques Used: Transduction, Mutagenesis, Cell Culture, Infection, Staining, Flow Cytometry, Produced, Enzyme-linked Immunosorbent Assay, Construct

Effect of SIVcol Nef on viral replication in  ex vivo  HLT and NF-κB or NF-AT activity. (A) Replication kinetics of HIV-1 constructs containing the indicated  nef  alleles in blocks of human lymphoid tissues  ex vivo . Panels A to C show mean values (±SEM) obtained using tissues from seven different donors. (B) Cumulative virus production over 15 days of infection by the indicated HIV-1 constructs (see panel A) relative to the replication of the HIV-1 construct containing the NA7  nef  allele (100%). (C) Levels of CD4+ T cell depletion in the tissue blocks (left) and cells that migrated in the gel foams (right) at the end of culture at 15 days post-infection. (D) HEK293T cells were cotransfected with a firefly luciferase reporter construct under the control of three NF-κB binding sites, a  Gaussia  luciferase construct for normalization, and expression vectors for a constitutively active mutant of IKKβ and the indicated Nef variants. Luciferase activity was determined 40 h post-transfection. The mean value of 9 transfections + SEM is shown. (E) Jurkat cells stably transfected with an NF-AT-dependent luciferase reporter gene were transduced with the indicated HIV-1 Nef-IRES-eGFP variants. The levels of luciferase activity were determined at 16 h post-stimulation. Shown are average values (+SD) derived from triplicate transductions relative to the  nef -defective control HIV-1 construct (100%). Similar results were obtained in two independent experiments. *, p
Figure Legend Snippet: Effect of SIVcol Nef on viral replication in ex vivo HLT and NF-κB or NF-AT activity. (A) Replication kinetics of HIV-1 constructs containing the indicated nef alleles in blocks of human lymphoid tissues ex vivo . Panels A to C show mean values (±SEM) obtained using tissues from seven different donors. (B) Cumulative virus production over 15 days of infection by the indicated HIV-1 constructs (see panel A) relative to the replication of the HIV-1 construct containing the NA7 nef allele (100%). (C) Levels of CD4+ T cell depletion in the tissue blocks (left) and cells that migrated in the gel foams (right) at the end of culture at 15 days post-infection. (D) HEK293T cells were cotransfected with a firefly luciferase reporter construct under the control of three NF-κB binding sites, a Gaussia luciferase construct for normalization, and expression vectors for a constitutively active mutant of IKKβ and the indicated Nef variants. Luciferase activity was determined 40 h post-transfection. The mean value of 9 transfections + SEM is shown. (E) Jurkat cells stably transfected with an NF-AT-dependent luciferase reporter gene were transduced with the indicated HIV-1 Nef-IRES-eGFP variants. The levels of luciferase activity were determined at 16 h post-stimulation. Shown are average values (+SD) derived from triplicate transductions relative to the nef -defective control HIV-1 construct (100%). Similar results were obtained in two independent experiments. *, p

Techniques Used: Ex Vivo, Activity Assay, Construct, Infection, Luciferase, Binding Assay, Expressing, Mutagenesis, Transfection, Stable Transfection, Transduction, Derivative Assay

13) Product Images from "High dose vitamin D exacerbates central nervous system autoimmunity by raising T-cell excitatory calcium"

Article Title: High dose vitamin D exacerbates central nervous system autoimmunity by raising T-cell excitatory calcium

Journal: Brain

doi: 10.1093/brain/awz190

Vitamin D and its metabolites inhibit activation of both human and murine CD4 + and CD8 + T cells. ( A , C and E ) Splenocytes were isolated from naïve wild-type mice receiving standard vitamin D diet. ( B , D and F ) Human PBMCs were isolated from healthy donors after Ficoll gradient centrifugation. ( A – F ) MACS purified murine or human T cells were CFSE labelled and incubated with increasing concentrations of ( A and B ) 25-(OH)-vitamin D, ( C and D ) 1,25-(OH) 2 -vitamin D or ( E and F ) cholecalciferol at 37°C. After 1 h, T cells were transferred to anti-CD3/anti-CD28 pre-coated wells and incubated for 48–72 h (murine T cells) or 96–120 h (human T cells). ( A – F ) T-cell proliferation was evaluated by CFSE dilution and stratified by division frequency as follows: few divisions (1–2; dark grey), intermediate divisions (3–4; medium grey) and many divisions (≥5; light grey). T-cell divisions are shown as mean ± SEM; representative plots of two independent experiments; n = 3.
Figure Legend Snippet: Vitamin D and its metabolites inhibit activation of both human and murine CD4 + and CD8 + T cells. ( A , C and E ) Splenocytes were isolated from naïve wild-type mice receiving standard vitamin D diet. ( B , D and F ) Human PBMCs were isolated from healthy donors after Ficoll gradient centrifugation. ( A – F ) MACS purified murine or human T cells were CFSE labelled and incubated with increasing concentrations of ( A and B ) 25-(OH)-vitamin D, ( C and D ) 1,25-(OH) 2 -vitamin D or ( E and F ) cholecalciferol at 37°C. After 1 h, T cells were transferred to anti-CD3/anti-CD28 pre-coated wells and incubated for 48–72 h (murine T cells) or 96–120 h (human T cells). ( A – F ) T-cell proliferation was evaluated by CFSE dilution and stratified by division frequency as follows: few divisions (1–2; dark grey), intermediate divisions (3–4; medium grey) and many divisions (≥5; light grey). T-cell divisions are shown as mean ± SEM; representative plots of two independent experiments; n = 3.

Techniques Used: Activation Assay, Isolation, Mouse Assay, Gradient Centrifugation, Magnetic Cell Separation, Purification, Incubation

Hypercalcaemia increases proliferation and encephalitogenic differentiation of both human and murine CD4 + and CD8 + T cells. ( A, C and E ) Splenocytes were isolated from naïve wild-type mice receiving standard vitamin D diet. ( B, D, F, G and H ) Human PBMCs were isolated from healthy donors after Ficoll gradient centrifugation. ( A and B ) MACS purified murine or human T cells were CFSE labelled and incubated with increasing calcium concentrations at 37°C. After 1 h, T cells were transferred to anti-CD3/anti-CD28 pre-coated wells and incubated for 48–72 h (murine T cells) or 96–120 h (human T cells). T-cell proliferation was evaluated by CFSE dilution and stratified by division frequency as follows: few divisions (1–2; dark grey), intermediate divisions (3–4; medium grey) and many divisions (≥5; light grey). T-cell divisions are shown as mean ± SEM; representative plots of two independent experiments; n = 3. MACS purified ( C ) murine or ( D ) human T cells were incubated with concentrations of ionized (free) calcium equivalent to concentrations measured in serum or culture medium. After 1 h, T cells were stained with Fluo-3 AM and Fura Red AM and calcium flux was evaluated by FACS. Representative calcium flux is shown left and area under the curve is depicted on the right (data given as mean ± SEM; ( C ) pooled plots from two independent experiments; n = 5; ( D ) pooled plots from three independent experiments; n = 8). MACS purified ( E ) murine or ( F ) human T cells were incubated with increasing calcium concentrations at 37°C. After 1 h, T cells were transferred to anti-CD3/anti-CD28 pre-coated wells and incubated for 1–3 h (murine T cells) or 3–20 h (human T cells). Total RNA was isolated, transcribed into cDNA and analysed by qPCR (data given as mean ± SEM; ( E ) pooled plots from two independent experiments; n = 4, ( F ) pooled plots from two independent experiments; n = 6–7). Number of CD4 + ( G ) and CD8 + ( H ) T cells in bottom chamber after 16 h migration over inflamed human BBB-ECs (modified Boyden chamber assay; left : total number of cells; right : number of cytokine-producing cells), following activation of T cells in the presence of various calcium concentrations. One million activated human T lymphocytes were seeded (data given as mean ± SEM; n = 6 different T-cell donors, two different BBB-EC preparations).
Figure Legend Snippet: Hypercalcaemia increases proliferation and encephalitogenic differentiation of both human and murine CD4 + and CD8 + T cells. ( A, C and E ) Splenocytes were isolated from naïve wild-type mice receiving standard vitamin D diet. ( B, D, F, G and H ) Human PBMCs were isolated from healthy donors after Ficoll gradient centrifugation. ( A and B ) MACS purified murine or human T cells were CFSE labelled and incubated with increasing calcium concentrations at 37°C. After 1 h, T cells were transferred to anti-CD3/anti-CD28 pre-coated wells and incubated for 48–72 h (murine T cells) or 96–120 h (human T cells). T-cell proliferation was evaluated by CFSE dilution and stratified by division frequency as follows: few divisions (1–2; dark grey), intermediate divisions (3–4; medium grey) and many divisions (≥5; light grey). T-cell divisions are shown as mean ± SEM; representative plots of two independent experiments; n = 3. MACS purified ( C ) murine or ( D ) human T cells were incubated with concentrations of ionized (free) calcium equivalent to concentrations measured in serum or culture medium. After 1 h, T cells were stained with Fluo-3 AM and Fura Red AM and calcium flux was evaluated by FACS. Representative calcium flux is shown left and area under the curve is depicted on the right (data given as mean ± SEM; ( C ) pooled plots from two independent experiments; n = 5; ( D ) pooled plots from three independent experiments; n = 8). MACS purified ( E ) murine or ( F ) human T cells were incubated with increasing calcium concentrations at 37°C. After 1 h, T cells were transferred to anti-CD3/anti-CD28 pre-coated wells and incubated for 1–3 h (murine T cells) or 3–20 h (human T cells). Total RNA was isolated, transcribed into cDNA and analysed by qPCR (data given as mean ± SEM; ( E ) pooled plots from two independent experiments; n = 4, ( F ) pooled plots from two independent experiments; n = 6–7). Number of CD4 + ( G ) and CD8 + ( H ) T cells in bottom chamber after 16 h migration over inflamed human BBB-ECs (modified Boyden chamber assay; left : total number of cells; right : number of cytokine-producing cells), following activation of T cells in the presence of various calcium concentrations. One million activated human T lymphocytes were seeded (data given as mean ± SEM; n = 6 different T-cell donors, two different BBB-EC preparations).

Techniques Used: Isolation, Mouse Assay, Gradient Centrifugation, Magnetic Cell Separation, Purification, Incubation, Staining, FACS, Real-time Polymerase Chain Reaction, Migration, Modification, Boyden Chamber Assay, Activation Assay

14) Product Images from "Obesity and Sex Affect the Immune Responses to Tick-Borne Encephalitis Booster Vaccination"

Article Title: Obesity and Sex Affect the Immune Responses to Tick-Borne Encephalitis Booster Vaccination

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.00860

Quantification of naïve and memory sub-populations of B lymphocytes and plasma blasts. Peripheral blood mononuclear cells were stained with fluorochrome-labeled mAbs and analyzed on a BD FACS Canto II flow cytometer. (A) Scatterplot of CD19 + B cells with expression of IgD and CD27 to distinguish naïve, un-switched, switched and double negative (DN) subsets. (B) Quantification of total CD19 + B-cells and of (B1) naïve, (B2) un-switched, and (B3) switched subset before (d0) and one week (d7) after TBE booster vaccination; line is arithmetic mean. (C) Plasma blasts (CD19 + /CD27 ++ /CD38 high ) as percentages of total CD19 + B cells before (d0) and 1 week (d7) after booster; line is arithmetic mean. ANOVA with linear contrasts; *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.
Figure Legend Snippet: Quantification of naïve and memory sub-populations of B lymphocytes and plasma blasts. Peripheral blood mononuclear cells were stained with fluorochrome-labeled mAbs and analyzed on a BD FACS Canto II flow cytometer. (A) Scatterplot of CD19 + B cells with expression of IgD and CD27 to distinguish naïve, un-switched, switched and double negative (DN) subsets. (B) Quantification of total CD19 + B-cells and of (B1) naïve, (B2) un-switched, and (B3) switched subset before (d0) and one week (d7) after TBE booster vaccination; line is arithmetic mean. (C) Plasma blasts (CD19 + /CD27 ++ /CD38 high ) as percentages of total CD19 + B cells before (d0) and 1 week (d7) after booster; line is arithmetic mean. ANOVA with linear contrasts; *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.

Techniques Used: Staining, Labeling, FACS, Flow Cytometry, Expressing

Quantification of circulating T follicular helper cells (cTfh) and cTfh1, cTfh2, and cTfh17 subsets. Peripheral blood mononuclear cells were stained with fluorochrome-labeled mAbs and analyzed on a BD FACS Canto II flow cytometer. (A) Scatterplot of total cTfh (CD3 + /CD4 + /CD45RA − /CXCR5 + ) with respective expression of CXCR3 and/or CCR6 to distinguish cTfh1, cTfh2, and cTfh17 subsets. CXCR3 + CCR6 − cell are cTfh1 cells; CXCR3 − CCR6 − are cTfh2 cells, and CXCR3 − CCR6 + cells are cTfh17 cells. (B) Quantification of cTfh (CD3 + /CD4 + /CD45RA − /CXCR5+) before (d0) and 1 week (d7) after booster vaccination as % of total CD4 + T cells. (C) cTfh1 and (D) cTfh17 cells as % of total cTfh before (d0) and 1 week (d7) after booster; line is arithmetic mean. (E) cTfh17 cells as % of total cTfh before TBE booster (d0) in females and males. Analysis of variance with linear contrasts; * p ≤ 0.05; *** p ≤ 0.05.
Figure Legend Snippet: Quantification of circulating T follicular helper cells (cTfh) and cTfh1, cTfh2, and cTfh17 subsets. Peripheral blood mononuclear cells were stained with fluorochrome-labeled mAbs and analyzed on a BD FACS Canto II flow cytometer. (A) Scatterplot of total cTfh (CD3 + /CD4 + /CD45RA − /CXCR5 + ) with respective expression of CXCR3 and/or CCR6 to distinguish cTfh1, cTfh2, and cTfh17 subsets. CXCR3 + CCR6 − cell are cTfh1 cells; CXCR3 − CCR6 − are cTfh2 cells, and CXCR3 − CCR6 + cells are cTfh17 cells. (B) Quantification of cTfh (CD3 + /CD4 + /CD45RA − /CXCR5+) before (d0) and 1 week (d7) after booster vaccination as % of total CD4 + T cells. (C) cTfh1 and (D) cTfh17 cells as % of total cTfh before (d0) and 1 week (d7) after booster; line is arithmetic mean. (E) cTfh17 cells as % of total cTfh before TBE booster (d0) in females and males. Analysis of variance with linear contrasts; * p ≤ 0.05; *** p ≤ 0.05.

Techniques Used: Staining, Labeling, FACS, Flow Cytometry, Expressing

Cytokine production of re-stimulated PBMCs. Interleukin 2 and IFN-γ concentrations were measured in PBMC culture supernatants with Luminex technology. Geometric mean (GM) concentrations (pg/mL) with 95% CI from PBMCs obtained before (d0) and 1 week (d7) after booster are shown; cytokine concentrations obtained in media-only cultures were subtracted. (A,B) Interleukin 2 and IFN-γ concentrations (pg/ml) from PBMC incubated with 0.48 μg/mL TBE antigen for 48 h, and (C) IL-2 concentrations (pg/mL) from PBMCs incubated with 1 μg/mL Staphylococcus Enterotoxin B for 48 h. ANOVA with linear contrasts; ** p ≤ 0.01, * p ≤ 0.05.
Figure Legend Snippet: Cytokine production of re-stimulated PBMCs. Interleukin 2 and IFN-γ concentrations were measured in PBMC culture supernatants with Luminex technology. Geometric mean (GM) concentrations (pg/mL) with 95% CI from PBMCs obtained before (d0) and 1 week (d7) after booster are shown; cytokine concentrations obtained in media-only cultures were subtracted. (A,B) Interleukin 2 and IFN-γ concentrations (pg/ml) from PBMC incubated with 0.48 μg/mL TBE antigen for 48 h, and (C) IL-2 concentrations (pg/mL) from PBMCs incubated with 1 μg/mL Staphylococcus Enterotoxin B for 48 h. ANOVA with linear contrasts; ** p ≤ 0.01, * p ≤ 0.05.

Techniques Used: Luminex, Incubation

15) Product Images from "Restriction of Equine Infectious Anemia Virus by Equine APOBEC3 Cytidine Deaminases ▿Restriction of Equine Infectious Anemia Virus by Equine APOBEC3 Cytidine Deaminases ▿ †"

Article Title: Restriction of Equine Infectious Anemia Virus by Equine APOBEC3 Cytidine Deaminases ▿Restriction of Equine Infectious Anemia Virus by Equine APOBEC3 Cytidine Deaminases ▿ †

Journal: Journal of Virology

doi: 10.1128/JVI.00015-09

Inhibition of EIAV by eqA3s is independent from dUTPase and S2. wt, ΔDU, and ΔS2 EIAV viruses were produced in the presence or absence of the indicated HA-tagged A3 expression vectors. Relative infectivity of equal amounts of the reporter viruses was determined by quantification of luciferase activity in CrFK cells (A) or EML-3C macrophage-like cells (B) at 3 days postinfection. cps, counts per second.
Figure Legend Snippet: Inhibition of EIAV by eqA3s is independent from dUTPase and S2. wt, ΔDU, and ΔS2 EIAV viruses were produced in the presence or absence of the indicated HA-tagged A3 expression vectors. Relative infectivity of equal amounts of the reporter viruses was determined by quantification of luciferase activity in CrFK cells (A) or EML-3C macrophage-like cells (B) at 3 days postinfection. cps, counts per second.

Techniques Used: Inhibition, Produced, Expressing, Infection, Luciferase, Activity Assay

16) Product Images from "The endogenous danger signals HSP70 and MICA cooperate in the activation of cytotoxic effector functions of NK cells"

Article Title: The endogenous danger signals HSP70 and MICA cooperate in the activation of cytotoxic effector functions of NK cells

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/j.1582-4934.2008.00677.x

Partial inhibition of NK cell cytotoxicity against SAHA-treated melanoma cells by soluble NKG2D and anti-MICA antibody. (A) The mean of specific lysis ± S.D. of triplicates of Ge-con target cells by NK cells cultured for 5 days with IL-2 (100 U/ml) plus TKD (2 μg/ml) is shown. The target cells were either cultured under standard conditions (co) or exposed to 10 μM SAHA for 20 hrs before the test. To inhibit the NKG2D dependent lysis a soluble human NKG2D-Fc fusion protein was added to the 51 Chromium release assay at a concentration of 3 μg/ml. The results of one individual out of three similar experiments are shown. (B) The mean of specific lysis ± S.D. of triplicates of Ge-con target cells by NK cells cultured for 5 days with IL-2 (100 U/ml) plus TKD (2 μg/ml) is shown. The target cells were either cultured under standard conditions (co) or exposed to 10 μM SAHA for 20 hrs before the test. To inhibit the NKG2D dependent lysis the anti-MICA mAb AMO1 or an isotype control (3 μg/ml) were added to the 51 Chromium release assay. The results of one individual out of three similar experiments are shown.
Figure Legend Snippet: Partial inhibition of NK cell cytotoxicity against SAHA-treated melanoma cells by soluble NKG2D and anti-MICA antibody. (A) The mean of specific lysis ± S.D. of triplicates of Ge-con target cells by NK cells cultured for 5 days with IL-2 (100 U/ml) plus TKD (2 μg/ml) is shown. The target cells were either cultured under standard conditions (co) or exposed to 10 μM SAHA for 20 hrs before the test. To inhibit the NKG2D dependent lysis a soluble human NKG2D-Fc fusion protein was added to the 51 Chromium release assay at a concentration of 3 μg/ml. The results of one individual out of three similar experiments are shown. (B) The mean of specific lysis ± S.D. of triplicates of Ge-con target cells by NK cells cultured for 5 days with IL-2 (100 U/ml) plus TKD (2 μg/ml) is shown. The target cells were either cultured under standard conditions (co) or exposed to 10 μM SAHA for 20 hrs before the test. To inhibit the NKG2D dependent lysis the anti-MICA mAb AMO1 or an isotype control (3 μg/ml) were added to the 51 Chromium release assay. The results of one individual out of three similar experiments are shown.

Techniques Used: Inhibition, Lysis, Cell Culture, Release Assay, Concentration Assay

17) Product Images from "Transient antibody targeting of CD45RC inhibits the development of graft-versus-host disease"

Article Title: Transient antibody targeting of CD45RC inhibits the development of graft-versus-host disease

Journal: Blood Advances

doi: 10.1182/bloodadvances.2020001688

Anti-human CD45RC ABIS-45RC treatment improves aGVHD survival in immune-humanized NSG mice. (A) Total human PBMCs or PBMCs depleted in CD45RC high or CD45RA high or preincubated with anti-CD45RC mAb were transferred in irradiated NSG mice to induce GVHD. Results are expressed in percentage of weight loss (left) and mice survival (right) (2 experiments). ** P
Figure Legend Snippet: Anti-human CD45RC ABIS-45RC treatment improves aGVHD survival in immune-humanized NSG mice. (A) Total human PBMCs or PBMCs depleted in CD45RC high or CD45RA high or preincubated with anti-CD45RC mAb were transferred in irradiated NSG mice to induce GVHD. Results are expressed in percentage of weight loss (left) and mice survival (right) (2 experiments). ** P

Techniques Used: Mouse Assay, Irradiation

ABIS-45RC chimeric anti-human CD45RC mAb induces cell death of CD45RC high T cells. (A) Expression levels of CD45RC using ABIS-45RC mAb on different human leukocyte subsets. Staining with ABIS-45RC was realized in whole blood (EDTA) from healthy volunteers after lysis of red blood cells before cytometry analysis. Cells were first gated on morphology, singlet cells, and live cells. Mean expression ± SEM of CD45RC high , CD45RC low , and CD45RC − on different cell types (n = 3) (1 experiment). (B) Human PBMCs incubated at 37°C with 10 µg/mL ABIS-45RC or isotype control mAbs were stained with annexin V and DAPI to analyze apoptosis. Results are expressed as relative proportion of annexin V + cells among CD3 + cells (left) or CD3 − cells (right) for 1 to 18 hours. A representative staining of ABIS-45RC on the different populations analyzed is shown (bottom). ** P
Figure Legend Snippet: ABIS-45RC chimeric anti-human CD45RC mAb induces cell death of CD45RC high T cells. (A) Expression levels of CD45RC using ABIS-45RC mAb on different human leukocyte subsets. Staining with ABIS-45RC was realized in whole blood (EDTA) from healthy volunteers after lysis of red blood cells before cytometry analysis. Cells were first gated on morphology, singlet cells, and live cells. Mean expression ± SEM of CD45RC high , CD45RC low , and CD45RC − on different cell types (n = 3) (1 experiment). (B) Human PBMCs incubated at 37°C with 10 µg/mL ABIS-45RC or isotype control mAbs were stained with annexin V and DAPI to analyze apoptosis. Results are expressed as relative proportion of annexin V + cells among CD3 + cells (left) or CD3 − cells (right) for 1 to 18 hours. A representative staining of ABIS-45RC on the different populations analyzed is shown (bottom). ** P

Techniques Used: Expressing, Staining, Lysis, Cytometry, Incubation

Anti-CD45RC mAb and rapamycin treatment induces tolerance in T - cell transferred Lew/BN F1 rats. (A) Control staining was realized on Lew (RT1 l ), BN (RT1 n ) and F1 Lew/BN (RT1 l/n ) PBMCs using anti-RT1 l and anti-RT1 n antibodies. (B) Top, Representative staining with anti-RT1 n and anti-RT1 l mAbs at different time points (days 7, 15, 22, and 50) on PBMCs of Lew/BN F1 rats injected with 2 × 10 7 Lew T cells and treated either with anti-CD45RC mAbs or isotype control mAbs and a suboptimal dose of rapamycin. Bottom, Results are expressed in mean of percentage of RT1 n - RT1 l+ cells among PBMCs ± SEM. Three independent experiments. (C) Absolute number of cells in spleen, blood (PBMC), BM, and lymph node (LN) were analyzed in Lew/BN F1 rats, long survivor rats transferred with either Lew/BN F1 T cells or Lew T cells and treated with anti-CD45RC mAbs and rapamycin. ** P
Figure Legend Snippet: Anti-CD45RC mAb and rapamycin treatment induces tolerance in T - cell transferred Lew/BN F1 rats. (A) Control staining was realized on Lew (RT1 l ), BN (RT1 n ) and F1 Lew/BN (RT1 l/n ) PBMCs using anti-RT1 l and anti-RT1 n antibodies. (B) Top, Representative staining with anti-RT1 n and anti-RT1 l mAbs at different time points (days 7, 15, 22, and 50) on PBMCs of Lew/BN F1 rats injected with 2 × 10 7 Lew T cells and treated either with anti-CD45RC mAbs or isotype control mAbs and a suboptimal dose of rapamycin. Bottom, Results are expressed in mean of percentage of RT1 n - RT1 l+ cells among PBMCs ± SEM. Three independent experiments. (C) Absolute number of cells in spleen, blood (PBMC), BM, and lymph node (LN) were analyzed in Lew/BN F1 rats, long survivor rats transferred with either Lew/BN F1 T cells or Lew T cells and treated with anti-CD45RC mAbs and rapamycin. ** P

Techniques Used: Staining, Injection

CD45RC expression is not altered before and after HSCT. (A) Expression level of CD45RC was analyzed on human T cells from PBMCs from healthy individuals or 88 patients between 15 and 65 years of age with de novo or secondary acute myeloid leukemia (AML) before (pregraft [PG]) hematopoietic stem cell transplantation (HSCT) or at month 3 (M3) and month 24 (M24) after HSCT. Mean expression ± SEM of CD45RC high , CD45RC low , CD45RC − at the different times analyzed. (B) Representative staining is shown. Cells were first gated on morphology, singlet cells, live cells, and CD3 + cells. FMO, Fluorescence Minus One control; FSC-A, forward scatter area; FSC-H, FSC height; SSC-A, side scatter area; SSC-H, SSC height.
Figure Legend Snippet: CD45RC expression is not altered before and after HSCT. (A) Expression level of CD45RC was analyzed on human T cells from PBMCs from healthy individuals or 88 patients between 15 and 65 years of age with de novo or secondary acute myeloid leukemia (AML) before (pregraft [PG]) hematopoietic stem cell transplantation (HSCT) or at month 3 (M3) and month 24 (M24) after HSCT. Mean expression ± SEM of CD45RC high , CD45RC low , CD45RC − at the different times analyzed. (B) Representative staining is shown. Cells were first gated on morphology, singlet cells, live cells, and CD3 + cells. FMO, Fluorescence Minus One control; FSC-A, forward scatter area; FSC-H, FSC height; SSC-A, side scatter area; SSC-H, SSC height.

Techniques Used: Expressing, Transplantation Assay, Staining, Fluorescence

ABIS-45RC treatment preserves elimination of tumor cells by GVT and memory antiviral immune responses. (A) NSG mice were injected subcutaneously with human MDA-MB-231 breast cancer cells and, when tumor growth was detectable (day 8), were transferred with total human PBMCs to induce GVT. Results are expressed in tumor volume (left) and mice survival (right) (2 experiments). * P
Figure Legend Snippet: ABIS-45RC treatment preserves elimination of tumor cells by GVT and memory antiviral immune responses. (A) NSG mice were injected subcutaneously with human MDA-MB-231 breast cancer cells and, when tumor growth was detectable (day 8), were transferred with total human PBMCs to induce GVT. Results are expressed in tumor volume (left) and mice survival (right) (2 experiments). * P

Techniques Used: Mouse Assay, Injection, Multiple Displacement Amplification

18) Product Images from "Species-specific differences in antagonism of APOBEC3 proteins by HIV-2 and SIVsmm Vif proteins"

Article Title: Species-specific differences in antagonism of APOBEC3 proteins by HIV-2 and SIVsmm Vif proteins

Journal: bioRxiv

doi: 10.1101/2020.09.08.287177

Replication of Vif mutant HIV-2 and SIVsmm IMCs in human CD4+ T cells. (A) Mean infectious virus yields measured by triplicate infection of TZM-bl reporter cells with normalized volumes of the supernatants from infected CD4+ T cell cultures derived from four different PBMC donors. ( B ) Infectious virus yields at 4 days post-infection. ( C ) Cumulative infectious virus production in PBMCs infected with the indicated SIVsmm constructs. Shown are AUCs for the replication kinetics as indicated in panel A but calculated for each of the four donors (indicated by a different symbol) individually. *, P
Figure Legend Snippet: Replication of Vif mutant HIV-2 and SIVsmm IMCs in human CD4+ T cells. (A) Mean infectious virus yields measured by triplicate infection of TZM-bl reporter cells with normalized volumes of the supernatants from infected CD4+ T cell cultures derived from four different PBMC donors. ( B ) Infectious virus yields at 4 days post-infection. ( C ) Cumulative infectious virus production in PBMCs infected with the indicated SIVsmm constructs. Shown are AUCs for the replication kinetics as indicated in panel A but calculated for each of the four donors (indicated by a different symbol) individually. *, P

Techniques Used: Mutagenesis, Infection, Derivative Assay, Construct

HIV-1, HIV-2 and SIVsmm differ significantly in IFN sensitivity. ( A ) Replication of HIV-1, HIV-2 and SIV proviral constructs in primary human CD4+ T cells. The results show mean infectious virus yields (n = 3) measured by infection of TZM-bl reporter cells with normalized volumes of the supernatants infected CD4+ T cell cultures derived from three different PBMC donors. ( B ) Mean infectious virus yield (±SEM) of the five HIV-1, five HIV-2 and six SIVsmm IMCs measured at the indicated days post-infection. ( C ) Replication of HIV-1, HIV-2 and SIV IMCs in CD4+ T cells infected as described in panel A the presence of IFN-α. ( D ) Mean infectious virus yields of HIV-1, HIV-2 and SIVsmm detected in the presence of IFN-α.
Figure Legend Snippet: HIV-1, HIV-2 and SIVsmm differ significantly in IFN sensitivity. ( A ) Replication of HIV-1, HIV-2 and SIV proviral constructs in primary human CD4+ T cells. The results show mean infectious virus yields (n = 3) measured by infection of TZM-bl reporter cells with normalized volumes of the supernatants infected CD4+ T cell cultures derived from three different PBMC donors. ( B ) Mean infectious virus yield (±SEM) of the five HIV-1, five HIV-2 and six SIVsmm IMCs measured at the indicated days post-infection. ( C ) Replication of HIV-1, HIV-2 and SIV IMCs in CD4+ T cells infected as described in panel A the presence of IFN-α. ( D ) Mean infectious virus yields of HIV-1, HIV-2 and SIVsmm detected in the presence of IFN-α.

Techniques Used: Construct, Infection, Derivative Assay

19) Product Images from "CD96 expression determines the inflammatory potential of IL-9–producing Th9 cells"

Article Title: CD96 expression determines the inflammatory potential of IL-9–producing Th9 cells

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

doi: 10.1073/pnas.1708329115

Single cell profiling identifies CD96 high and CD96 low subsets of Th9 cells. ( A – D ) Single cell gene expression analysis of CD44 + alloreactive T cells sorted from nonpolarized (w/o; n = 100), Th1 ( n = 67), and Th9 ( n = 113) polarized BALB/c cocultures. See Table S2 for a list of all genes measured. ( A ) Principal component analysis of gene expression in indicated Th subsets. ( B ) Nonsupervised hierarchical clustering analysis (Pearson’s correlation) comparing gene expression in Th subsets. Analysis includes all genes with detectable expression. ( C ) Box plots of most differentially expressed genes. Bonferroni-corrected ANOVA P values: Il9 ( P = 1.4 × 10 −126 ), Il2ra ( P = 1.4 × 10 −65 ), Cd83 ( P = 7.1 × 10 −64 ), Il4ra ( P = 1.2 × 10 −52 ), Gata3 ( P = 3.6 × 10 −47 ), Il1r1 ( P = 5.5 × 10 −47 ), Ccr4 ( P = 2.2 × 10 −46 ), and Cd28 ( P = 2.3 × 10 −43 ). ( D ) Nonsupervised hierarchical clustering analysis (complete linkage method) of gene expression in CD44 + T cells sorted from alloreactive BALB/c Th9 cultures. Displayed genes contributed to clustering. Frame indicates a cluster of Th9 cells characterized by high Cd96 expression. ( E ) Contour plots of surface CD96 expression in CD44 + polarized alloreactive BALB/c T cells, including fluorescence minus one (FMO) staining control (Ctrl). ( F ) Comparison of CD96 expression levels in IL-9 + and IFN-γ + human CD4 + T cells. PBMCs from blood of 10 healthy individuals were stimulated ex vivo with Staphylococcus enterotoxin B (SEB) for 24 h. A one-tailed Wilcoxon matched-pairs signed rank test was applied. gMFI, geometric mean fluorescence intensity.
Figure Legend Snippet: Single cell profiling identifies CD96 high and CD96 low subsets of Th9 cells. ( A – D ) Single cell gene expression analysis of CD44 + alloreactive T cells sorted from nonpolarized (w/o; n = 100), Th1 ( n = 67), and Th9 ( n = 113) polarized BALB/c cocultures. See Table S2 for a list of all genes measured. ( A ) Principal component analysis of gene expression in indicated Th subsets. ( B ) Nonsupervised hierarchical clustering analysis (Pearson’s correlation) comparing gene expression in Th subsets. Analysis includes all genes with detectable expression. ( C ) Box plots of most differentially expressed genes. Bonferroni-corrected ANOVA P values: Il9 ( P = 1.4 × 10 −126 ), Il2ra ( P = 1.4 × 10 −65 ), Cd83 ( P = 7.1 × 10 −64 ), Il4ra ( P = 1.2 × 10 −52 ), Gata3 ( P = 3.6 × 10 −47 ), Il1r1 ( P = 5.5 × 10 −47 ), Ccr4 ( P = 2.2 × 10 −46 ), and Cd28 ( P = 2.3 × 10 −43 ). ( D ) Nonsupervised hierarchical clustering analysis (complete linkage method) of gene expression in CD44 + T cells sorted from alloreactive BALB/c Th9 cultures. Displayed genes contributed to clustering. Frame indicates a cluster of Th9 cells characterized by high Cd96 expression. ( E ) Contour plots of surface CD96 expression in CD44 + polarized alloreactive BALB/c T cells, including fluorescence minus one (FMO) staining control (Ctrl). ( F ) Comparison of CD96 expression levels in IL-9 + and IFN-γ + human CD4 + T cells. PBMCs from blood of 10 healthy individuals were stimulated ex vivo with Staphylococcus enterotoxin B (SEB) for 24 h. A one-tailed Wilcoxon matched-pairs signed rank test was applied. gMFI, geometric mean fluorescence intensity.

Techniques Used: Expressing, Fluorescence, Staining, Ex Vivo, One-tailed Test

20) Product Images from "Accumulation and therapeutic modulation of 6-sulfo LacNAc+ dendritic cells in multiple sclerosis"

Article Title: Accumulation and therapeutic modulation of 6-sulfo LacNAc+ dendritic cells in multiple sclerosis

Journal: Neurology® Neuroimmunology & Neuroinflammation

doi: 10.1212/NXI.0000000000000033

Impact of short-term MP administration on frequency and phenotype of slanDCs and monocytes in the blood of patients with MS The percentages of (A) 6-sulfo LacNAc + dendritic cells (slanDCs) or (B) monocytes in peripheral blood mononuclear cells (PBMCs) derived from 11 patients with relapsing-remitting multiple sclerosis (RRMS) before and during methylprednisolone (MP) treatment are presented. (C–F) PBMCs derived from the blood of 11 patients with RRMS before and during MP treatment were stimulated with lipopolysaccharide (LPS) or R848. The percentages of tumor necrosis factor (TNF)-α–expressing slanDCs (C) or monocytes (D) as well as CD150-expressing slanDCs (E) or monocytes (F) in PBMCs are demonstrated. Values represent the mean ± SEM of results. The upper row of asterisks indicates a statistically significant difference between R848-activated slanDCs or monocytes on day 1 or day 2 compared to day 0. The lower row of asterisks indicates a statistically significant difference between LPS-activated slanDCs or monocytes on day 1 or day 2 compared to day 0. Asterisks indicate statistical significance (* p
Figure Legend Snippet: Impact of short-term MP administration on frequency and phenotype of slanDCs and monocytes in the blood of patients with MS The percentages of (A) 6-sulfo LacNAc + dendritic cells (slanDCs) or (B) monocytes in peripheral blood mononuclear cells (PBMCs) derived from 11 patients with relapsing-remitting multiple sclerosis (RRMS) before and during methylprednisolone (MP) treatment are presented. (C–F) PBMCs derived from the blood of 11 patients with RRMS before and during MP treatment were stimulated with lipopolysaccharide (LPS) or R848. The percentages of tumor necrosis factor (TNF)-α–expressing slanDCs (C) or monocytes (D) as well as CD150-expressing slanDCs (E) or monocytes (F) in PBMCs are demonstrated. Values represent the mean ± SEM of results. The upper row of asterisks indicates a statistically significant difference between R848-activated slanDCs or monocytes on day 1 or day 2 compared to day 0. The lower row of asterisks indicates a statistically significant difference between LPS-activated slanDCs or monocytes on day 1 or day 2 compared to day 0. Asterisks indicate statistical significance (* p

Techniques Used: Mass Spectrometry, Derivative Assay, Expressing

Impact of IFN-β on relevant proinflammatory capabilities of slanDCs The percentages of (A) 6-sulfo LacNAc + dendritic cells (slanDCs) or (B) monocytes in peripheral blood mononuclear cells (PBMCs) d erived from blood of 30 patients with relapsing-remitting multiple sclerosis (RRMS) treated with interferon (IFN)-β, glatiramer acetate (GA), or natalizumab (NA); untreated patients with RRMS (de novo); and healthy donors (CTRL) are depicted. In addition, the percentages of (A) slanDCs or (B) monocytes in PBMCs derived from blood of 4 IFN-β-treated patients with RRMS who developed neutralizing anti-IFN-β antibodies (Abs) are shown. Horizontal bars show the mean values. After immunomagnetic cell sorting, slanDCs were stimulated with lipopolysaccharide (LPS) and cultured with or without IFN-β for 24 hours. Supernatant concentrations of (C) tumor necrosis factor (TNF)-α, (D) interleukin (IL)-1β, (E) IL-6, and (F) IL-12p70 were evaluated by ELISA. IFN-β pretreated slanDCs were co-cultured with allogeneic (G) CD4 + or (H) CD8 + T cells. After 4 days 3 H-thymidine incorporation was examined to assess T-cell proliferation. Findings of 3 different analyses are presented as mean ± SEM of triplicate determinations. (I) IFN-β-treated slanDCs were co-cultured with allogeneic naive CD45RA + CD4 + T cells and stimulated with LPS. After 8 days of cell culture, expression of IFN-γ or IL-4 of CD4 + T cells was evaluated by flow cytometry. Three independent analyses presented similar results. Results of one representative donor are presented. Asterisks indicate statistical significance (* p
Figure Legend Snippet: Impact of IFN-β on relevant proinflammatory capabilities of slanDCs The percentages of (A) 6-sulfo LacNAc + dendritic cells (slanDCs) or (B) monocytes in peripheral blood mononuclear cells (PBMCs) d erived from blood of 30 patients with relapsing-remitting multiple sclerosis (RRMS) treated with interferon (IFN)-β, glatiramer acetate (GA), or natalizumab (NA); untreated patients with RRMS (de novo); and healthy donors (CTRL) are depicted. In addition, the percentages of (A) slanDCs or (B) monocytes in PBMCs derived from blood of 4 IFN-β-treated patients with RRMS who developed neutralizing anti-IFN-β antibodies (Abs) are shown. Horizontal bars show the mean values. After immunomagnetic cell sorting, slanDCs were stimulated with lipopolysaccharide (LPS) and cultured with or without IFN-β for 24 hours. Supernatant concentrations of (C) tumor necrosis factor (TNF)-α, (D) interleukin (IL)-1β, (E) IL-6, and (F) IL-12p70 were evaluated by ELISA. IFN-β pretreated slanDCs were co-cultured with allogeneic (G) CD4 + or (H) CD8 + T cells. After 4 days 3 H-thymidine incorporation was examined to assess T-cell proliferation. Findings of 3 different analyses are presented as mean ± SEM of triplicate determinations. (I) IFN-β-treated slanDCs were co-cultured with allogeneic naive CD45RA + CD4 + T cells and stimulated with LPS. After 8 days of cell culture, expression of IFN-γ or IL-4 of CD4 + T cells was evaluated by flow cytometry. Three independent analyses presented similar results. Results of one representative donor are presented. Asterisks indicate statistical significance (* p

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

21) Product Images from "Age-related differences in humoral and cellular immune responses after primary immunisation: indications for stratified vaccination schedules"

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

Journal: Scientific Reports

doi: 10.1038/s41598-018-28111-8

Vaccine-specific cytokine production. ( a ) IL-2, ( b ) IFN-γ and ( c ) IL-10 production of PBMC after 48 hour stimulation in vitro with JE virus antigen and TBE virus antigen (TBEV), respectively, or incubation with just medium. Cytokine levels were measured in supernatants. ( d ) JE-specific IFN-γ/IL-10 ratio after subtraction of medium values. Statistical analysis by General Linear Model with log-transformed values. Individual comparisons by linear contrasts. * p
Figure Legend Snippet: Vaccine-specific cytokine production. ( a ) IL-2, ( b ) IFN-γ and ( c ) IL-10 production of PBMC after 48 hour stimulation in vitro with JE virus antigen and TBE virus antigen (TBEV), respectively, or incubation with just medium. Cytokine levels were measured in supernatants. ( d ) JE-specific IFN-γ/IL-10 ratio after subtraction of medium values. Statistical analysis by General Linear Model with log-transformed values. Individual comparisons by linear contrasts. * p

Techniques Used: In Vitro, Incubation, Transformation Assay

22) Product Images from "Indoleamine 2,3-dioxygenase provides adaptive resistance to immune checkpoint inhibitors in hepatocellular carcinoma"

Article Title: Indoleamine 2,3-dioxygenase provides adaptive resistance to immune checkpoint inhibitors in hepatocellular carcinoma

Journal: Cancer immunology, immunotherapy : CII

doi: 10.1007/s00262-018-2190-4

In vitro stimulation of human HCC tumor cells showed variable levels of IDO1 and IDO2 expression. Hep3B (a) or HepG2 (b) tumor cells (500,000 cells) cultured with 1000u IFN-γ or a cytokine rich supernatant isolated from 5×10 6 stimulated human PBMCs for 24 hours. Induction of IDO1 and IDO2 was measured by RT-qPCR. *: P
Figure Legend Snippet: In vitro stimulation of human HCC tumor cells showed variable levels of IDO1 and IDO2 expression. Hep3B (a) or HepG2 (b) tumor cells (500,000 cells) cultured with 1000u IFN-γ or a cytokine rich supernatant isolated from 5×10 6 stimulated human PBMCs for 24 hours. Induction of IDO1 and IDO2 was measured by RT-qPCR. *: P

Techniques Used: In Vitro, Expressing, Cell Culture, Isolation, Quantitative RT-PCR

23) Product Images from "Compartment-specific distribution of human intestinal innate lymphoid cells is altered in HIV patients under effective therapy"

Article Title: Compartment-specific distribution of human intestinal innate lymphoid cells is altered in HIV patients under effective therapy

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006373

Compartment-specific distribution and function of intestinal ILCs in HIV(-) individuals. (A) Frequency of ILCs (black dots, left panel) and cNK cells (white dots; right panel) expressed as percentage of CD45(+) lymphocytes in the different parts of the human GI tract (ILCs: oesophagus, n = 10; stomach n = 15; duodenum, n = 21; ileum, n = 13; colon: n = 13; cNK: oesophagus, n = 12; stomach n = 15; duodenum, n = 21; ileum, n = 14; colon: n = 13; grey columns) in comparison to peripheral blood (n = 16; white columns). (B) Distribution of CD103(+)ILC1 (upper left panel), CD127(+)ILC1 (upper right panel), ILC2 (lower left panel), and ILC3 (lower right panel) in the human intestinal tract (black columns) and the circulating blood (white columns). (C) Composition of CD127(+)ILC1 (blue), intraepithelial ILC1 (red), ILC2 (green) and ILC3 (violet) in the peripheral blood and the different segments of the gastrointestinal tract. (D) Distribution of NCR(-) (left panel) and NCR(+) ILC3 (right panel) in the peripheral blood and the different segments of the gastrointestinal tract. (E) Percentage comparison between NCR(-) and NCR(+) ILC3 subsets in those compartments. For functional characterisation ILCs derived from PBMC or biopsy samples (oesophagus, stomach, duodenum, Ileum and colon) obtained from HIV(-) individuals, were stimulated with PMA/ionomycin and then tested for IFN-γ production in CD103(+)ILC1 (F) , CD127(+)ILC1 (G) ,or IL-22 production in ILC3 (H) , respectively. * p≤0.05; ** p≤0.01; *** p≤0.001 (FDR-adjusted P values, parametric ANOVA).
Figure Legend Snippet: Compartment-specific distribution and function of intestinal ILCs in HIV(-) individuals. (A) Frequency of ILCs (black dots, left panel) and cNK cells (white dots; right panel) expressed as percentage of CD45(+) lymphocytes in the different parts of the human GI tract (ILCs: oesophagus, n = 10; stomach n = 15; duodenum, n = 21; ileum, n = 13; colon: n = 13; cNK: oesophagus, n = 12; stomach n = 15; duodenum, n = 21; ileum, n = 14; colon: n = 13; grey columns) in comparison to peripheral blood (n = 16; white columns). (B) Distribution of CD103(+)ILC1 (upper left panel), CD127(+)ILC1 (upper right panel), ILC2 (lower left panel), and ILC3 (lower right panel) in the human intestinal tract (black columns) and the circulating blood (white columns). (C) Composition of CD127(+)ILC1 (blue), intraepithelial ILC1 (red), ILC2 (green) and ILC3 (violet) in the peripheral blood and the different segments of the gastrointestinal tract. (D) Distribution of NCR(-) (left panel) and NCR(+) ILC3 (right panel) in the peripheral blood and the different segments of the gastrointestinal tract. (E) Percentage comparison between NCR(-) and NCR(+) ILC3 subsets in those compartments. For functional characterisation ILCs derived from PBMC or biopsy samples (oesophagus, stomach, duodenum, Ileum and colon) obtained from HIV(-) individuals, were stimulated with PMA/ionomycin and then tested for IFN-γ production in CD103(+)ILC1 (F) , CD127(+)ILC1 (G) ,or IL-22 production in ILC3 (H) , respectively. * p≤0.05; ** p≤0.01; *** p≤0.001 (FDR-adjusted P values, parametric ANOVA).

Techniques Used: Functional Assay, Derivative Assay

24) Product Images from "Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis"

Article Title: Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis

Journal: Cancers

doi: 10.3390/cancers12082085

In vitro lactic acidosis affects IFN-α production by pDCs. pDCs purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 plus lactic acid (10 mM; 15 mM; 20 mM) ( n = 7; ( A )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5) ( n = 7; ( B )) for 24 h. pDCs were stimulated with R848 for 2 h. Intracellular IFN-α was analyzed by flow cytometry. Aligned dot plot graphs show the percentages of IFN-α + pDCs evaluated on BDCA-2 + /CD123 + cells. Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p
Figure Legend Snippet: In vitro lactic acidosis affects IFN-α production by pDCs. pDCs purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 plus lactic acid (10 mM; 15 mM; 20 mM) ( n = 7; ( A )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5) ( n = 7; ( B )) for 24 h. pDCs were stimulated with R848 for 2 h. Intracellular IFN-α was analyzed by flow cytometry. Aligned dot plot graphs show the percentages of IFN-α + pDCs evaluated on BDCA-2 + /CD123 + cells. Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p

Techniques Used: In Vitro, Purification, Cell Culture, Flow Cytometry

Lactic acidosis affects the viability of pDCs and T cells. pDCs and T cells purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS plus lactic Acid (10 mM; 15 mM; 20 mM) ( n = 7 ( A – C ); n = 6, ( D – F )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5 ( n = 6 ( G – I ); n = 7, ( J – L )) for 24 h. IL-3 was added to pDCs’ culture. The cellular viability was analyzed by annexin V/SYTOX AADvanced staining in flow cytometry. Aligned dot plot graphs show the percentages of dead ( A , D , G , J ), early apoptotic ( B , E , H , K ), and late apoptotic or necrotic cells ( C , F , I , L ). Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p
Figure Legend Snippet: Lactic acidosis affects the viability of pDCs and T cells. pDCs and T cells purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS plus lactic Acid (10 mM; 15 mM; 20 mM) ( n = 7 ( A – C ); n = 6, ( D – F )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5 ( n = 6 ( G – I ); n = 7, ( J – L )) for 24 h. IL-3 was added to pDCs’ culture. The cellular viability was analyzed by annexin V/SYTOX AADvanced staining in flow cytometry. Aligned dot plot graphs show the percentages of dead ( A , D , G , J ), early apoptotic ( B , E , H , K ), and late apoptotic or necrotic cells ( C , F , I , L ). Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p

Techniques Used: Purification, Cell Culture, Staining, Flow Cytometry

Frequency of peripheral blood immune cells and IFN-α + pDCs in BRAF V600+ MM patients over therapy administration. Cell counts ( A ) and flow cytometry ( B , C ) analysis were performed on whole blood from BRAF V600+ MM patients before therapy initiation (T0; n = 16), after 30 days (T1; n = 12), and after 120 days (T2; n = 9) from therapy administration ( A – C ). Total PBMCs isolated from peripheral blood of MM patients were cultured in RPMI 1640 medium and stimulated with R848 for 2 h ( D ). IFN-α was analyzed by intracellular flow cytometry staining ( D ). Before–after graphs illustrate the frequency of lymphocytes ( A ), pDCs ( B ), and mDCs ( C ) on total PBMCs, and the frequency of IFN-α + pDCs on BDCA-2 + /CD123 + cells ( D ) for each subject. Bold black lines represent the median values. The statistical significance was calculated by Wilcoxon signed-rank test. * p
Figure Legend Snippet: Frequency of peripheral blood immune cells and IFN-α + pDCs in BRAF V600+ MM patients over therapy administration. Cell counts ( A ) and flow cytometry ( B , C ) analysis were performed on whole blood from BRAF V600+ MM patients before therapy initiation (T0; n = 16), after 30 days (T1; n = 12), and after 120 days (T2; n = 9) from therapy administration ( A – C ). Total PBMCs isolated from peripheral blood of MM patients were cultured in RPMI 1640 medium and stimulated with R848 for 2 h ( D ). IFN-α was analyzed by intracellular flow cytometry staining ( D ). Before–after graphs illustrate the frequency of lymphocytes ( A ), pDCs ( B ), and mDCs ( C ) on total PBMCs, and the frequency of IFN-α + pDCs on BDCA-2 + /CD123 + cells ( D ) for each subject. Bold black lines represent the median values. The statistical significance was calculated by Wilcoxon signed-rank test. * p

Techniques Used: Flow Cytometry, Isolation, Cell Culture, Staining

Frequency of interferon alpha (IFN-α) and CXCL10-producing plasmacytoid dendritic cells (pDCs) in chemo-naïve MM patients and HD. Representative dot plots of R848-stimulated IFN-α + and CXCL10 + pDC subsets obtained from HD and MM patients are shown ( A ). PBMCs were isolated from peripheral blood of HD ( n = 25) and MM patients ( n = 29). Total PBMCs were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 and stimulated with R848 or IMQ for 2 h ( B , D ) and 6 h ( C , E ), and with CpG-ODN 2216 for 6 h ( B – E ). IFN-α ( B , D ) and CXCL10 ( C , E ) were analyzed by intracellular flow cytometry staining. Scatter dot plot graphs illustrate the percentages of positive pDCs evaluated on BDCA-2 + /CD123 + cells. Subgroup analysis of the MM cohort illustrating the frequency of IFN-α + and CXCL10 + pDCs in M1a-c categories ( D , E ). Median and IQR are shown in ( B , C ). Mean and SD are shown in ( D , E ). The statistical significance was calculated by Wilcoxon–Mann–Whitney test ( B , C ) and by a Student’s t -test ( D , E ). * p
Figure Legend Snippet: Frequency of interferon alpha (IFN-α) and CXCL10-producing plasmacytoid dendritic cells (pDCs) in chemo-naïve MM patients and HD. Representative dot plots of R848-stimulated IFN-α + and CXCL10 + pDC subsets obtained from HD and MM patients are shown ( A ). PBMCs were isolated from peripheral blood of HD ( n = 25) and MM patients ( n = 29). Total PBMCs were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 and stimulated with R848 or IMQ for 2 h ( B , D ) and 6 h ( C , E ), and with CpG-ODN 2216 for 6 h ( B – E ). IFN-α ( B , D ) and CXCL10 ( C , E ) were analyzed by intracellular flow cytometry staining. Scatter dot plot graphs illustrate the percentages of positive pDCs evaluated on BDCA-2 + /CD123 + cells. Subgroup analysis of the MM cohort illustrating the frequency of IFN-α + and CXCL10 + pDCs in M1a-c categories ( D , E ). Median and IQR are shown in ( B , C ). Mean and SD are shown in ( D , E ). The statistical significance was calculated by Wilcoxon–Mann–Whitney test ( B , C ) and by a Student’s t -test ( D , E ). * p

Techniques Used: Isolation, Cell Culture, Flow Cytometry, Staining, MANN-WHITNEY

25) Product Images from "Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis"

Article Title: Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis

Journal: Cancers

doi: 10.3390/cancers12082085

In vitro lactic acidosis affects IFN-α production by pDCs. pDCs purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 plus lactic acid (10 mM; 15 mM; 20 mM) ( n = 7; ( A )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5) ( n = 7; ( B )) for 24 h. pDCs were stimulated with R848 for 2 h. Intracellular IFN-α was analyzed by flow cytometry. Aligned dot plot graphs show the percentages of IFN-α + pDCs evaluated on BDCA-2 + /CD123 + cells. Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p
Figure Legend Snippet: In vitro lactic acidosis affects IFN-α production by pDCs. pDCs purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 plus lactic acid (10 mM; 15 mM; 20 mM) ( n = 7; ( A )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5) ( n = 7; ( B )) for 24 h. pDCs were stimulated with R848 for 2 h. Intracellular IFN-α was analyzed by flow cytometry. Aligned dot plot graphs show the percentages of IFN-α + pDCs evaluated on BDCA-2 + /CD123 + cells. Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p

Techniques Used: In Vitro, Purification, Cell Culture, Flow Cytometry

Lactic acidosis affects the viability of pDCs and T cells. pDCs and T cells purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS plus lactic Acid (10 mM; 15 mM; 20 mM) ( n = 7 ( A – C ); n = 6, ( D – F )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5 ( n = 6 ( G – I ); n = 7, ( J – L )) for 24 h. IL-3 was added to pDCs’ culture. The cellular viability was analyzed by annexin V/SYTOX AADvanced staining in flow cytometry. Aligned dot plot graphs show the percentages of dead ( A , D , G , J ), early apoptotic ( B , E , H , K ), and late apoptotic or necrotic cells ( C , F , I , L ). Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p
Figure Legend Snippet: Lactic acidosis affects the viability of pDCs and T cells. pDCs and T cells purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS plus lactic Acid (10 mM; 15 mM; 20 mM) ( n = 7 ( A – C ); n = 6, ( D – F )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5 ( n = 6 ( G – I ); n = 7, ( J – L )) for 24 h. IL-3 was added to pDCs’ culture. The cellular viability was analyzed by annexin V/SYTOX AADvanced staining in flow cytometry. Aligned dot plot graphs show the percentages of dead ( A , D , G , J ), early apoptotic ( B , E , H , K ), and late apoptotic or necrotic cells ( C , F , I , L ). Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p

Techniques Used: Purification, Cell Culture, Staining, Flow Cytometry

Frequency of interferon alpha (IFN-α) and CXCL10-producing plasmacytoid dendritic cells (pDCs) in chemo-naïve MM patients and HD. Representative dot plots of R848-stimulated IFN-α + and CXCL10 + pDC subsets obtained from HD and MM patients are shown ( A ). PBMCs were isolated from peripheral blood of HD ( n = 25) and MM patients ( n = 29). Total PBMCs were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 and stimulated with R848 or IMQ for 2 h ( B , D ) and 6 h ( C , E ), and with CpG-ODN 2216 for 6 h ( B – E ). IFN-α ( B , D ) and CXCL10 ( C , E ) were analyzed by intracellular flow cytometry staining. Scatter dot plot graphs illustrate the percentages of positive pDCs evaluated on BDCA-2 + /CD123 + cells. Subgroup analysis of the MM cohort illustrating the frequency of IFN-α + and CXCL10 + pDCs in M1a-c categories ( D , E ). Median and IQR are shown in ( B , C ). Mean and SD are shown in ( D , E ). The statistical significance was calculated by Wilcoxon–Mann–Whitney test ( B , C ) and by a Student’s t -test ( D , E ). * p
Figure Legend Snippet: Frequency of interferon alpha (IFN-α) and CXCL10-producing plasmacytoid dendritic cells (pDCs) in chemo-naïve MM patients and HD. Representative dot plots of R848-stimulated IFN-α + and CXCL10 + pDC subsets obtained from HD and MM patients are shown ( A ). PBMCs were isolated from peripheral blood of HD ( n = 25) and MM patients ( n = 29). Total PBMCs were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 and stimulated with R848 or IMQ for 2 h ( B , D ) and 6 h ( C , E ), and with CpG-ODN 2216 for 6 h ( B – E ). IFN-α ( B , D ) and CXCL10 ( C , E ) were analyzed by intracellular flow cytometry staining. Scatter dot plot graphs illustrate the percentages of positive pDCs evaluated on BDCA-2 + /CD123 + cells. Subgroup analysis of the MM cohort illustrating the frequency of IFN-α + and CXCL10 + pDCs in M1a-c categories ( D , E ). Median and IQR are shown in ( B , C ). Mean and SD are shown in ( D , E ). The statistical significance was calculated by Wilcoxon–Mann–Whitney test ( B , C ) and by a Student’s t -test ( D , E ). * p

Techniques Used: Isolation, Cell Culture, Flow Cytometry, Staining, MANN-WHITNEY

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In Vitro:

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Isolation:

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Centrifugation:

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Modification:

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Gradient Centrifugation:

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    Biochrom peripheral blood mononuclear cells pbmc
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    Biochrom human primary cd4 t cell culture human peripheral blood mononuclear cells pbmcs
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    Biochrom pbmc isolation human peripheral blood mononuclear cells pbmcs
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    Biochrom murine leucocytes human pbmcs
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    miR-20b expression in T cells and in PBMC from natalizumab-associated progressive multifocal leukoencephalopathy (PML). (A) PBMC were isolated using a Ficoll gradient and CD4+ T cells were sorted with magnetic beads ( n = 9; randomly selected patients of our initial longitudinal cohort consisting of 17 patients). miR-20b expression (qPCR) was measured in the CD4+ and CD4-negative fraction and compared using t- test. (B) CD4+ cells were activated by plate-bound anti-CD3-antibody and soluble anti-CD28-antibody without further cytokines, termed “Th0”, and with IL-1 (10 ng/mL), IL-6 (10 ng/mL), IL-23 (10 ng/mL) and TGF- β (5 ng/mL), termed “Th17”, as well as freshly isolated CD4+ T cells, termed “fresh”. miR-20b levels were measured by qPCR. (C) miR-20b levels in PBMC from natalizumab-treated patients ( n = 9, left bar) were compared to miR-20b levels in two PBMC samples from patients with natalizumab-associated PML (each patient one bar).

    Journal: Annals of Clinical and Translational Neurology

    Article Title: Natalizumab restores aberrant miRNA expression profile in multiple sclerosis and reveals a critical role for miR-20b

    doi: 10.1002/acn3.152

    Figure Lengend Snippet: miR-20b expression in T cells and in PBMC from natalizumab-associated progressive multifocal leukoencephalopathy (PML). (A) PBMC were isolated using a Ficoll gradient and CD4+ T cells were sorted with magnetic beads ( n = 9; randomly selected patients of our initial longitudinal cohort consisting of 17 patients). miR-20b expression (qPCR) was measured in the CD4+ and CD4-negative fraction and compared using t- test. (B) CD4+ cells were activated by plate-bound anti-CD3-antibody and soluble anti-CD28-antibody without further cytokines, termed “Th0”, and with IL-1 (10 ng/mL), IL-6 (10 ng/mL), IL-23 (10 ng/mL) and TGF- β (5 ng/mL), termed “Th17”, as well as freshly isolated CD4+ T cells, termed “fresh”. miR-20b levels were measured by qPCR. (C) miR-20b levels in PBMC from natalizumab-treated patients ( n = 9, left bar) were compared to miR-20b levels in two PBMC samples from patients with natalizumab-associated PML (each patient one bar).

    Article Snippet: Peripheral blood mononuclear cells (PBMC) were isolated from whole blood by Ficoll (Biochrom, Berlin, Germany) gradient and CD4+ cells were isolated by magnetic bead separation using STEMCELL EasySep Human CD4+ T Cell Enrichment Kit according to the manufacturer’s instructions.

    Techniques: Expressing, Isolation, Magnetic Beads, Real-time Polymerase Chain Reaction

    Differential miRNA expression 3h, 6h and 24h after IFN I stimulation of human primary CD4+ T cells. A) Heat map for differentially expressed miRNAs. The heatmap represents relative expression values for a non-redundant collection of differentially expressed miRNAs (adjusted p-value

    Journal: bioRxiv

    Article Title: Impaired miRNA degradation by post-transcriptional addition of 3’ cytosine and adenine in T cell activation

    doi: 10.1101/2020.08.19.257816

    Figure Lengend Snippet: Differential miRNA expression 3h, 6h and 24h after IFN I stimulation of human primary CD4+ T cells. A) Heat map for differentially expressed miRNAs. The heatmap represents relative expression values for a non-redundant collection of differentially expressed miRNAs (adjusted p-value

    Article Snippet: Human primary CD4 T cell culture Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats, obtained from healthy donors, by separation on Biocoll Separating Solution (Biochrom, L6115) according to standard procedures.

    Techniques: Expressing

    Differential miRNA expression 3h, 6h and 24h after αCD3αCD28 stimulation of human primary CD4+ T cells. A) Heat map for differentially expressed miRNAs. The heatmap represents relative expression values for a non-redundant collection of differentially expressed miRNAs (adjusted p-value

    Journal: bioRxiv

    Article Title: Impaired miRNA degradation by post-transcriptional addition of 3’ cytosine and adenine in T cell activation

    doi: 10.1101/2020.08.19.257816

    Figure Lengend Snippet: Differential miRNA expression 3h, 6h and 24h after αCD3αCD28 stimulation of human primary CD4+ T cells. A) Heat map for differentially expressed miRNAs. The heatmap represents relative expression values for a non-redundant collection of differentially expressed miRNAs (adjusted p-value

    Article Snippet: Human primary CD4 T cell culture Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats, obtained from healthy donors, by separation on Biocoll Separating Solution (Biochrom, L6115) according to standard procedures.

    Techniques: Expressing

    Expression of uridylated RNA degrading enzymes (Dis3L2 and Eri1) and terminal uridyl transferases (TUT4 y TUT7) upon CD4+ T cell activation. Western blot analysis of protein expression in human primary CD4+ T cell stimulated with αCD3αCD28, assessing Dis3L2 (A), Eri1 (B), TUT4 (C) and TUT7 (D). Fold increase compared to non-stimulation was represented for each donor to observe evolution upon activation (left panel) and using group median and interquartile range with whiskers ranging from minimum to maximum values (middle panel). Right panels include two examples for each protein to highlight inter-donor variability in upregulation kinetics. Band intensities were normalized to ERMs values and relativized to unstimulated conditions. Statistical analysis: Kruskal-Wallis test, Dunn’s multiple comparisons test [* p-value

    Journal: bioRxiv

    Article Title: Impaired miRNA degradation by post-transcriptional addition of 3’ cytosine and adenine in T cell activation

    doi: 10.1101/2020.08.19.257816

    Figure Lengend Snippet: Expression of uridylated RNA degrading enzymes (Dis3L2 and Eri1) and terminal uridyl transferases (TUT4 y TUT7) upon CD4+ T cell activation. Western blot analysis of protein expression in human primary CD4+ T cell stimulated with αCD3αCD28, assessing Dis3L2 (A), Eri1 (B), TUT4 (C) and TUT7 (D). Fold increase compared to non-stimulation was represented for each donor to observe evolution upon activation (left panel) and using group median and interquartile range with whiskers ranging from minimum to maximum values (middle panel). Right panels include two examples for each protein to highlight inter-donor variability in upregulation kinetics. Band intensities were normalized to ERMs values and relativized to unstimulated conditions. Statistical analysis: Kruskal-Wallis test, Dunn’s multiple comparisons test [* p-value

    Article Snippet: Human primary CD4 T cell culture Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats, obtained from healthy donors, by separation on Biocoll Separating Solution (Biochrom, L6115) according to standard procedures.

    Techniques: Expressing, Activation Assay, Western Blot

    Tenocytes produce IL-6 upon stimulation. Cytokines were measured in media from αCD3αCD28 stimulated PBMCs (stim. media) (see Fig. 1a ) using the Legendplex TM human inflammation panel. After tenocytes were cultured for 3 days with either the control media from unstimulated PBMC (unstim. media + tenocytes; dotted line) or stimulation media (stim. media + tenocytes), cytokines were measured in the supernatants. The levels of IL-6 ( a ), IL-8 ( b ), MCP-1 ( c ) and IFNγ ( d ) are shown for the tenocytes cultured with stimulated media compared to the stimulated media without tenocytes present. The median levels for tenocytes incubated with the unstimulated media are shown next to the dotted line at that level on a–c , and IFNγ was undetectable. Samples were treated following the manufacturer’s instructions and measured by flow cytometry. Analysis was done using Legendplex software. Data are presented as the median with the interquartile range with an n = 5 for stimulated media alone and n = 10 for stimulated media + tenocytes, and are considered significantly different when *p

    Journal: Scientific Reports

    Article Title: New insights into tenocyte-immune cell interplay in an in vitro model of inflammation

    doi: 10.1038/s41598-017-09875-x

    Figure Lengend Snippet: Tenocytes produce IL-6 upon stimulation. Cytokines were measured in media from αCD3αCD28 stimulated PBMCs (stim. media) (see Fig. 1a ) using the Legendplex TM human inflammation panel. After tenocytes were cultured for 3 days with either the control media from unstimulated PBMC (unstim. media + tenocytes; dotted line) or stimulation media (stim. media + tenocytes), cytokines were measured in the supernatants. The levels of IL-6 ( a ), IL-8 ( b ), MCP-1 ( c ) and IFNγ ( d ) are shown for the tenocytes cultured with stimulated media compared to the stimulated media without tenocytes present. The median levels for tenocytes incubated with the unstimulated media are shown next to the dotted line at that level on a–c , and IFNγ was undetectable. Samples were treated following the manufacturer’s instructions and measured by flow cytometry. Analysis was done using Legendplex software. Data are presented as the median with the interquartile range with an n = 5 for stimulated media alone and n = 10 for stimulated media + tenocytes, and are considered significantly different when *p

    Article Snippet: PBMC isolation Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats using a Biocoll density gradient (Biochrom, Berlin, Germany) with centrifugation at 800 g for 30 minutes at room temperature with no brake.

    Techniques: Cell Culture, Incubation, Flow Cytometry, Cytometry, Software

    Potential role of cross-talk between tenocytes and macrophages in an inflammatory milieu in tendinopathy. The scheme illustrates the effects of inflammatory conditions on human tenocytes alone and during their interplay with human macrophages on surface marker expression and cytokine release patterns. A mixed cytokine stimulation originating from αCD3αCD28 activated human PBMCs including all subsets (T cells, B cells, NK cells and monocytes (Mo)) induces the up-regulation of HLA-molecules (HLA-DR, HLA-ABC) and adhesion molecules (ICAM-1, VCAM-1) on tenocytes and enhances their IL-6 secretion. After co-culturing monocyte-derived macrophages (Mϕ) with pre-stimulated tenocytes, the macrophages display a mixed M1/M2 macrophage phenotype with enhanced expression of CD80 (characteristic for M1-Mϕ), but reduced HLA-DR (characteristic for M2-Mϕ). As yet unknown soluble factors lead to higher levels of IL-6, IL-8 and MCP-1 secretion. We hypothesize, that this interaction might contribute to a disturbed resolution of the immune response after tendon injury leading to chronic tendinopathies.

    Journal: Scientific Reports

    Article Title: New insights into tenocyte-immune cell interplay in an in vitro model of inflammation

    doi: 10.1038/s41598-017-09875-x

    Figure Lengend Snippet: Potential role of cross-talk between tenocytes and macrophages in an inflammatory milieu in tendinopathy. The scheme illustrates the effects of inflammatory conditions on human tenocytes alone and during their interplay with human macrophages on surface marker expression and cytokine release patterns. A mixed cytokine stimulation originating from αCD3αCD28 activated human PBMCs including all subsets (T cells, B cells, NK cells and monocytes (Mo)) induces the up-regulation of HLA-molecules (HLA-DR, HLA-ABC) and adhesion molecules (ICAM-1, VCAM-1) on tenocytes and enhances their IL-6 secretion. After co-culturing monocyte-derived macrophages (Mϕ) with pre-stimulated tenocytes, the macrophages display a mixed M1/M2 macrophage phenotype with enhanced expression of CD80 (characteristic for M1-Mϕ), but reduced HLA-DR (characteristic for M2-Mϕ). As yet unknown soluble factors lead to higher levels of IL-6, IL-8 and MCP-1 secretion. We hypothesize, that this interaction might contribute to a disturbed resolution of the immune response after tendon injury leading to chronic tendinopathies.

    Article Snippet: PBMC isolation Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats using a Biocoll density gradient (Biochrom, Berlin, Germany) with centrifugation at 800 g for 30 minutes at room temperature with no brake.

    Techniques: Marker, Expressing, Derivative Assay

    Pro-inflammatory stimulation media increases the size and granularity of tenocytes, and changes their surface marker expression. Following incubation for 3 days with either the control media from unstimulated PBMC (unstim. media) or the pro-inflammatory stimulation media from αCD3αCD28 stimulated PBMCs (stim. media) ( a ), supernatants were collected for later cytokine evaluation before the tenocytes were stained with a panel of human-specific antibodies to characteristic surface markers and analyzed by flow cytometry. Tenocytes included in the viable gate with unstim. media ( b ) or with the stim. media ( c ) are shown here with representative flow cytometry density plots. The mean fluorescence intensity (MFI) of forward scatter area (FSC-A) and sidewards scatter area (SSC-A) indicating cell size and granularity are shown ( d ). The expression of the adhesion molecules ICAM-1 (CD54) and VCAM-1 (CD106), as well as the HLA class I molecule HLA-ABC and the HLA class II molecule HLA-DR ( e ) and CD90 (Thy-1) ( f ) are also shown after incubation with unstimulated and stimulated media. Data are presented as the median of n = 10 with the interquartile range and are considered significantly different when *p

    Journal: Scientific Reports

    Article Title: New insights into tenocyte-immune cell interplay in an in vitro model of inflammation

    doi: 10.1038/s41598-017-09875-x

    Figure Lengend Snippet: Pro-inflammatory stimulation media increases the size and granularity of tenocytes, and changes their surface marker expression. Following incubation for 3 days with either the control media from unstimulated PBMC (unstim. media) or the pro-inflammatory stimulation media from αCD3αCD28 stimulated PBMCs (stim. media) ( a ), supernatants were collected for later cytokine evaluation before the tenocytes were stained with a panel of human-specific antibodies to characteristic surface markers and analyzed by flow cytometry. Tenocytes included in the viable gate with unstim. media ( b ) or with the stim. media ( c ) are shown here with representative flow cytometry density plots. The mean fluorescence intensity (MFI) of forward scatter area (FSC-A) and sidewards scatter area (SSC-A) indicating cell size and granularity are shown ( d ). The expression of the adhesion molecules ICAM-1 (CD54) and VCAM-1 (CD106), as well as the HLA class I molecule HLA-ABC and the HLA class II molecule HLA-DR ( e ) and CD90 (Thy-1) ( f ) are also shown after incubation with unstimulated and stimulated media. Data are presented as the median of n = 10 with the interquartile range and are considered significantly different when *p

    Article Snippet: PBMC isolation Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats using a Biocoll density gradient (Biochrom, Berlin, Germany) with centrifugation at 800 g for 30 minutes at room temperature with no brake.

    Techniques: Marker, Expressing, Incubation, Staining, Flow Cytometry, Cytometry, Fluorescence

    Vitamin D and its metabolites inhibit activation of both human and murine CD4 + and CD8 + T cells. ( A , C and E ) Splenocytes were isolated from naïve wild-type mice receiving standard vitamin D diet. ( B , D and F ) Human PBMCs were isolated from healthy donors after Ficoll gradient centrifugation. ( A – F ) MACS purified murine or human T cells were CFSE labelled and incubated with increasing concentrations of ( A and B ) 25-(OH)-vitamin D, ( C and D ) 1,25-(OH) 2 -vitamin D or ( E and F ) cholecalciferol at 37°C. After 1 h, T cells were transferred to anti-CD3/anti-CD28 pre-coated wells and incubated for 48–72 h (murine T cells) or 96–120 h (human T cells). ( A – F ) T-cell proliferation was evaluated by CFSE dilution and stratified by division frequency as follows: few divisions (1–2; dark grey), intermediate divisions (3–4; medium grey) and many divisions (≥5; light grey). T-cell divisions are shown as mean ± SEM; representative plots of two independent experiments; n = 3.

    Journal: Brain

    Article Title: High dose vitamin D exacerbates central nervous system autoimmunity by raising T-cell excitatory calcium

    doi: 10.1093/brain/awz190

    Figure Lengend Snippet: Vitamin D and its metabolites inhibit activation of both human and murine CD4 + and CD8 + T cells. ( A , C and E ) Splenocytes were isolated from naïve wild-type mice receiving standard vitamin D diet. ( B , D and F ) Human PBMCs were isolated from healthy donors after Ficoll gradient centrifugation. ( A – F ) MACS purified murine or human T cells were CFSE labelled and incubated with increasing concentrations of ( A and B ) 25-(OH)-vitamin D, ( C and D ) 1,25-(OH) 2 -vitamin D or ( E and F ) cholecalciferol at 37°C. After 1 h, T cells were transferred to anti-CD3/anti-CD28 pre-coated wells and incubated for 48–72 h (murine T cells) or 96–120 h (human T cells). ( A – F ) T-cell proliferation was evaluated by CFSE dilution and stratified by division frequency as follows: few divisions (1–2; dark grey), intermediate divisions (3–4; medium grey) and many divisions (≥5; light grey). T-cell divisions are shown as mean ± SEM; representative plots of two independent experiments; n = 3.

    Article Snippet: Isolation of human and murine leucocytes Human PBMCs from healthy donors were isolated after Ficoll gradient centrifugation (Biochrom).

    Techniques: Activation Assay, Isolation, Mouse Assay, Gradient Centrifugation, Magnetic Cell Separation, Purification, Incubation

    Hypercalcaemia increases proliferation and encephalitogenic differentiation of both human and murine CD4 + and CD8 + T cells. ( A, C and E ) Splenocytes were isolated from naïve wild-type mice receiving standard vitamin D diet. ( B, D, F, G and H ) Human PBMCs were isolated from healthy donors after Ficoll gradient centrifugation. ( A and B ) MACS purified murine or human T cells were CFSE labelled and incubated with increasing calcium concentrations at 37°C. After 1 h, T cells were transferred to anti-CD3/anti-CD28 pre-coated wells and incubated for 48–72 h (murine T cells) or 96–120 h (human T cells). T-cell proliferation was evaluated by CFSE dilution and stratified by division frequency as follows: few divisions (1–2; dark grey), intermediate divisions (3–4; medium grey) and many divisions (≥5; light grey). T-cell divisions are shown as mean ± SEM; representative plots of two independent experiments; n = 3. MACS purified ( C ) murine or ( D ) human T cells were incubated with concentrations of ionized (free) calcium equivalent to concentrations measured in serum or culture medium. After 1 h, T cells were stained with Fluo-3 AM and Fura Red AM and calcium flux was evaluated by FACS. Representative calcium flux is shown left and area under the curve is depicted on the right (data given as mean ± SEM; ( C ) pooled plots from two independent experiments; n = 5; ( D ) pooled plots from three independent experiments; n = 8). MACS purified ( E ) murine or ( F ) human T cells were incubated with increasing calcium concentrations at 37°C. After 1 h, T cells were transferred to anti-CD3/anti-CD28 pre-coated wells and incubated for 1–3 h (murine T cells) or 3–20 h (human T cells). Total RNA was isolated, transcribed into cDNA and analysed by qPCR (data given as mean ± SEM; ( E ) pooled plots from two independent experiments; n = 4, ( F ) pooled plots from two independent experiments; n = 6–7). Number of CD4 + ( G ) and CD8 + ( H ) T cells in bottom chamber after 16 h migration over inflamed human BBB-ECs (modified Boyden chamber assay; left : total number of cells; right : number of cytokine-producing cells), following activation of T cells in the presence of various calcium concentrations. One million activated human T lymphocytes were seeded (data given as mean ± SEM; n = 6 different T-cell donors, two different BBB-EC preparations).

    Journal: Brain

    Article Title: High dose vitamin D exacerbates central nervous system autoimmunity by raising T-cell excitatory calcium

    doi: 10.1093/brain/awz190

    Figure Lengend Snippet: Hypercalcaemia increases proliferation and encephalitogenic differentiation of both human and murine CD4 + and CD8 + T cells. ( A, C and E ) Splenocytes were isolated from naïve wild-type mice receiving standard vitamin D diet. ( B, D, F, G and H ) Human PBMCs were isolated from healthy donors after Ficoll gradient centrifugation. ( A and B ) MACS purified murine or human T cells were CFSE labelled and incubated with increasing calcium concentrations at 37°C. After 1 h, T cells were transferred to anti-CD3/anti-CD28 pre-coated wells and incubated for 48–72 h (murine T cells) or 96–120 h (human T cells). T-cell proliferation was evaluated by CFSE dilution and stratified by division frequency as follows: few divisions (1–2; dark grey), intermediate divisions (3–4; medium grey) and many divisions (≥5; light grey). T-cell divisions are shown as mean ± SEM; representative plots of two independent experiments; n = 3. MACS purified ( C ) murine or ( D ) human T cells were incubated with concentrations of ionized (free) calcium equivalent to concentrations measured in serum or culture medium. After 1 h, T cells were stained with Fluo-3 AM and Fura Red AM and calcium flux was evaluated by FACS. Representative calcium flux is shown left and area under the curve is depicted on the right (data given as mean ± SEM; ( C ) pooled plots from two independent experiments; n = 5; ( D ) pooled plots from three independent experiments; n = 8). MACS purified ( E ) murine or ( F ) human T cells were incubated with increasing calcium concentrations at 37°C. After 1 h, T cells were transferred to anti-CD3/anti-CD28 pre-coated wells and incubated for 1–3 h (murine T cells) or 3–20 h (human T cells). Total RNA was isolated, transcribed into cDNA and analysed by qPCR (data given as mean ± SEM; ( E ) pooled plots from two independent experiments; n = 4, ( F ) pooled plots from two independent experiments; n = 6–7). Number of CD4 + ( G ) and CD8 + ( H ) T cells in bottom chamber after 16 h migration over inflamed human BBB-ECs (modified Boyden chamber assay; left : total number of cells; right : number of cytokine-producing cells), following activation of T cells in the presence of various calcium concentrations. One million activated human T lymphocytes were seeded (data given as mean ± SEM; n = 6 different T-cell donors, two different BBB-EC preparations).

    Article Snippet: Isolation of human and murine leucocytes Human PBMCs from healthy donors were isolated after Ficoll gradient centrifugation (Biochrom).

    Techniques: Isolation, Mouse Assay, Gradient Centrifugation, Magnetic Cell Separation, Purification, Incubation, Staining, FACS, Real-time Polymerase Chain Reaction, Migration, Modification, Boyden Chamber Assay, Activation Assay