easysep rosettesep human cd4 t cell isolation kit  (STEMCELL Technologies Inc)

Journal: Immunity

Article Title: Diapedesis-induced integrin signalling via LFA-1 facilitates tissue immunity by inducing intrinsic complement C3 expression in immune cells

doi: 10.1016/j.immuni.2020.02.006

Figure Lengend Snippet: (A-C) Significantly enriched canonical pathways by GSEA comparing transcriptomes of human CD4+ (A) and CD8+ T cells (B) isolated from lung versus spleen (data from GSE94964) and monocytes and macrophages (C) isolated from tissues versus blood (data from GSE117970). Left, pathways ranked by significance (false-discovery rate q-values), with complement pathways highlighted in red and integrin pathways in yellow. Right, GSEA plots for the top complement pathways in A-C, respectively. (D-F) Expression of C3 mRNA in CD4+ (D) and CD8+ (E) T cells isolated from lung versus spleen (data from GSE94964) and monocytes and macrophages (F) isolated from tissues versus blood (data from GSE117970). (G) Schematic depicting the methicillin-resistant Staphylococcus aureus (MRSA) ear infection model. Wild type (WT) or C3-Td Tomato reporter mice (C3-TdT) were infected in the ear followed by intravital microscopy and blood and organ harvest at day 7 post-infection. (H) Still capture of an intravital imaging movie (see Movie S1) of an MRSA-infected ear section of a WT (left) and C3-TdT reporter mouse (right) at day 7 post-infection. Bar, 30 μm. (I) Representative FACS analysis (top) and cumulative data (below) showing Td Tomato reporter activity in CD4+ (n=7) and CD8+ (n=5) T cells of WT and C3-TdT mice from different tissues at day 7. (J) Representative FACS analysis (left) and cumulative data (right) showing Td Tomato reporter activity in tissue macrophages of WT and C3-TdT mice from the site of infection at day 7 (n=5). I-J show cumulative data from 3–5 experiments. Bars show mean + SEM. * p<0.05, ** p <0.01, ***p < 0.001, ****p < 0.0001. See also Figure S1 and Table S1.

Article Snippet: Human CD4 + and CD8 + T cell Purification and In Vitro Activation Human bulk CD4 + or CD8 + T cells were isolated from PBMCs (obtained from freshly drawn blood after centrifugation using Lymphoprep separation medium (Corning, Vienna, VA) using either the MACS Human CD8 + T cell Isolation Kit (130–096-495) from Miltenyi Biotech, (Bergisch Gladbach, Germany), the Negative Selection EasySep CD4 T cell kit (17951) or RosetteSep Human CD4 Cell Isolation Kit (15062) from Stemcell Technologies (Vancouver, Canada) according to the manufacturers’ instructions.

Techniques: Isolation, Expressing, Infection, Intravital Microscopy, Imaging, Activity Assay

Journal: Immunity

Article Title: Diapedesis-induced integrin signalling via LFA-1 facilitates tissue immunity by inducing intrinsic complement C3 expression in immune cells

doi: 10.1016/j.immuni.2020.02.006

Figure Lengend Snippet: (A) Schematic representation of the in vitro transmigration assay utilizing a trans-well system with HUVEC cells and sorted naive and memory CD4+ T cells. (B-D) Transmigration (B) of healthy donor naïve and memory CD4+ T cells across a trans-well system in the presence and absence of IL-1β (induces ICAM-1 expression by HUVEC cells), with and without DEL-1 (a specific inhibitor of LFA-1/ICAM-1 interaction) and expression of C3 (C) and IFNG (D) mRNA by transmigrated cells. Cumulative data from n=6–9 independent experiments. Bars show mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005. See also Figure S3.

Article Snippet: Human CD4 + and CD8 + T cell Purification and In Vitro Activation Human bulk CD4 + or CD8 + T cells were isolated from PBMCs (obtained from freshly drawn blood after centrifugation using Lymphoprep separation medium (Corning, Vienna, VA) using either the MACS Human CD8 + T cell Isolation Kit (130–096-495) from Miltenyi Biotech, (Bergisch Gladbach, Germany), the Negative Selection EasySep CD4 T cell kit (17951) or RosetteSep Human CD4 Cell Isolation Kit (15062) from Stemcell Technologies (Vancouver, Canada) according to the manufacturers’ instructions.

Techniques: In Vitro, Transmigration Assay, Expressing

Journal: Immunity

Article Title: Diapedesis-induced integrin signalling via LFA-1 facilitates tissue immunity by inducing intrinsic complement C3 expression in immune cells

doi: 10.1016/j.immuni.2020.02.006

Figure Lengend Snippet: (A) Schematic depicting the integrins and selectins (with respective binding partners in parentheses) involved in rolling, adhesion and transmigration assessed for C3-dependent Th1 induction. (B) Reporter activity of splenic CD4+ T cells from the C3-Td tomato reporter mouse activated with antibodies to CD3 or CD3+CD28 with or without increasing concentrations of immobilized ICAM-1 for seven days and C3-Td Tomato signal measured by flow cytometry. Shown are cumulative data from n=5 independent experiments. (C) C3 mRNA (above) and protein (below) expression over time in healthy donor naive and memory CD4+ T cells cultured in the presence or absence of ICAM-1 alone. Cumulative data (mean ± SEM; n=3 independent experiments); both the C3 mRNA and protein expression curves are higher for both memory and naive cells with ICAM-1 compared to without ICAM-1 (p<0.001 for mRNA expression in both cell types; p<0.0001 for C3 protein in naive T cells and p<0.01 for C3 protein in memory T cells). (D-E) Confocal images for C3 mRNA (using the PrimeFlow assay) and IFN-γ protein (D) and flow cytometry histograms for C3 mRNA (PrimeFlow assay) (E) in bulk CD4+ T cells activated for 36h in the presence and absence of ICAM-1. Shown are representative examples of four independent experiments (n=4). Size bar in ‘D’, 2 μm. Bars show mean + SEM throughout. *p < 0.05, **p < 0.01, ****p < 0.0001. See also Figure S2.

Article Snippet: Human CD4 + and CD8 + T cell Purification and In Vitro Activation Human bulk CD4 + or CD8 + T cells were isolated from PBMCs (obtained from freshly drawn blood after centrifugation using Lymphoprep separation medium (Corning, Vienna, VA) using either the MACS Human CD8 + T cell Isolation Kit (130–096-495) from Miltenyi Biotech, (Bergisch Gladbach, Germany), the Negative Selection EasySep CD4 T cell kit (17951) or RosetteSep Human CD4 Cell Isolation Kit (15062) from Stemcell Technologies (Vancouver, Canada) according to the manufacturers’ instructions.

Techniques: Binding Assay, Transmigration Assay, Activity Assay, Flow Cytometry, Expressing, Cell Culture

Journal: Immunity

Article Title: Diapedesis-induced integrin signalling via LFA-1 facilitates tissue immunity by inducing intrinsic complement C3 expression in immune cells

doi: 10.1016/j.immuni.2020.02.006

Figure Lengend Snippet: (A) IFN-γ secretion (left) and intracellular C3a generation (right) by healthy donor CD4+ T cells activated as shown in the presence and absence of ICAM-1 with and without a cell-permeable cathepsin L inhibitor (CTSLi) for 72h (n=4 independent experiments). (B) Representative flow cytometry plots from n=4 independent experiments showing C3 mRNA and the mTOR down-stream target S6 kinase phosphorylated at ser235/ser236 (p-S6) in healthy donor CD4+ T cells activated as shown in the presence or absence of ICAM-1 for 36h. (C) Extracellular acidification rate (ECAR, a measure of glycolysis) and oxygen consumption rate (OCR, a measure of oxidative phosphorylation) in healthy donor naive CD4+ T cells activated as shown in the presence or absence of ICAM-1 with and without a CTSL inhibitor for 36h. Shown are representative ECAR (above) and OCR (middle) graphs together with cumulative data of the maximal respiration and glycolysis (below) from n=3 independent experiments. NA, non-activated; FCCP, p-trifluoromethoxyphenyl hydrazone; OM, oligomycin; ROT, rotenone. Bars show mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. See also Figure S4.

Article Snippet: Human CD4 + and CD8 + T cell Purification and In Vitro Activation Human bulk CD4 + or CD8 + T cells were isolated from PBMCs (obtained from freshly drawn blood after centrifugation using Lymphoprep separation medium (Corning, Vienna, VA) using either the MACS Human CD8 + T cell Isolation Kit (130–096-495) from Miltenyi Biotech, (Bergisch Gladbach, Germany), the Negative Selection EasySep CD4 T cell kit (17951) or RosetteSep Human CD4 Cell Isolation Kit (15062) from Stemcell Technologies (Vancouver, Canada) according to the manufacturers’ instructions.

Techniques: Flow Cytometry, Phospho-proteomics

Journal: Immunity

Article Title: Diapedesis-induced integrin signalling via LFA-1 facilitates tissue immunity by inducing intrinsic complement C3 expression in immune cells

doi: 10.1016/j.immuni.2020.02.006

Figure Lengend Snippet: (A-D) C3 mRNA expression in naive (A and B) and memory (C and D) CD4+ T cells activated as indicated in the presence or absence of ICAM-1 with and without a cell-permeable AP-1 inhibitor (AP-1 inh.) or DMSO carrier control at 72h post activation. Shown are representative flow cytometry histograms of (A and C) and cumulative data from n=5 (B) and n=4 (D) independent experiments. NA, non-activated. Error bars represent mean + SEM. **p < 0.01 ***, p < 0.005, ****p < 0.001. See also Figure S5.

Article Snippet: Human CD4 + and CD8 + T cell Purification and In Vitro Activation Human bulk CD4 + or CD8 + T cells were isolated from PBMCs (obtained from freshly drawn blood after centrifugation using Lymphoprep separation medium (Corning, Vienna, VA) using either the MACS Human CD8 + T cell Isolation Kit (130–096-495) from Miltenyi Biotech, (Bergisch Gladbach, Germany), the Negative Selection EasySep CD4 T cell kit (17951) or RosetteSep Human CD4 Cell Isolation Kit (15062) from Stemcell Technologies (Vancouver, Canada) according to the manufacturers’ instructions.

Techniques: Expressing, Control, Activation Assay, Flow Cytometry

Journal: Immunity

Article Title: Diapedesis-induced integrin signalling via LFA-1 facilitates tissue immunity by inducing intrinsic complement C3 expression in immune cells

doi: 10.1016/j.immuni.2020.02.006

Figure Lengend Snippet: (A) Steady-state expression of C3 and IFNG mRNA in paired CD4+ T cells drawn from blood and synovial fluid of pediatric patients with juvenile idiopathic oligo-arthritis (n=4; Table S2). Bars show mean + SEM. (B) IFN-γ secretion by paired CD4+ T cells drawn from blood and synovial fluid of two patients with rheumatoid arthritis (RA) activated in vitro with antibodies against CD3 with, or without, ICAM-1. (C-D) C3 mRNA expression by synovial T cells of patients with either oligo-arthritis (OA), non-inflamed RA or inflamed RA derived from an independent dataset (Zhang et al., 2019) (C) and receiver operating characteristic curves showing performance of C3 and IFNG mRNA expression in synovial T cells as biomarkers to distinguish inflamed from un-inflamed RA (D). (E) LFA-1 expression (top), C3 mRNA (middle) and IFN-γ secretion (bottom) by peripheral blood CD4+ T cells from patients with LFA-1 mutations (LAD-1 disease) (see Table S3) and age- and sex-matched controls activated in vitro as shown with, and without ICAM-1 for 72h. Data are from three patients, two of whom donated twice, and five controls. Bars represent mean of duplicate measurements per subject. (F) regression lines showing correlation between IFN-γ and LFA-1 expression (left) and between C3 mRNA and LFA-1 expression (right) from primary data in (E). 95% confidence intervals of the regression line are shown and individual donors are marked. (G) IFN-γ secretion by peripheral blood CD4+ T cells from three patients with LAD-1 disease after transduction with control adenovirus or adenovirus encoding C3a, electroporation with mRNA encoding GFP or C3a or electroporation with BSA or C3H2O protein, as indicated. Cells were activated with anti-CD3+CD46+ICAM-1 after transduction or electroporation. Each patient has been labelled, two of whom donated three times, and the over-expression method shown by color-coding. *p<0.05. See also Figure S6 and Tables S2 and S3.

Article Snippet: Human CD4 + and CD8 + T cell Purification and In Vitro Activation Human bulk CD4 + or CD8 + T cells were isolated from PBMCs (obtained from freshly drawn blood after centrifugation using Lymphoprep separation medium (Corning, Vienna, VA) using either the MACS Human CD8 + T cell Isolation Kit (130–096-495) from Miltenyi Biotech, (Bergisch Gladbach, Germany), the Negative Selection EasySep CD4 T cell kit (17951) or RosetteSep Human CD4 Cell Isolation Kit (15062) from Stemcell Technologies (Vancouver, Canada) according to the manufacturers’ instructions.

Techniques: Expressing, In Vitro, Derivative Assay, Transduction, Control, Electroporation, Over Expression

Journal: Immunity

Article Title: Diapedesis-induced integrin signalling via LFA-1 facilitates tissue immunity by inducing intrinsic complement C3 expression in immune cells

doi: 10.1016/j.immuni.2020.02.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human CD4 + and CD8 + T cell Purification and In Vitro Activation Human bulk CD4 + or CD8 + T cells were isolated from PBMCs (obtained from freshly drawn blood after centrifugation using Lymphoprep separation medium (Corning, Vienna, VA) using either the MACS Human CD8 + T cell Isolation Kit (130–096-495) from Miltenyi Biotech, (Bergisch Gladbach, Germany), the Negative Selection EasySep CD4 T cell kit (17951) or RosetteSep Human CD4 Cell Isolation Kit (15062) from Stemcell Technologies (Vancouver, Canada) according to the manufacturers’ instructions.

Techniques: Virus, Generated, Control, Isolation, Recombinant, Purification, Staining, Diagnostic Assay, Cell Isolation, Enzyme-linked Immunosorbent Assay, Labeling, Gene Expression, Reverse Transcription, Synthesized, Software, Transfection, Imaging, Flow Cytometry, Microscopy, Western Blot

Journal: Immunity

Article Title: Diapedesis-induced integrin signalling via LFA-1 facilitates tissue immunity by inducing intrinsic complement C3 expression in immune cells

doi: 10.1016/j.immuni.2020.02.006

Figure Lengend Snippet: (A-C) Significantly enriched canonical pathways by GSEA comparing transcriptomes of human CD4+ (A) and CD8+ T cells (B) isolated from lung versus spleen (data from GSE94964) and monocytes and macrophages (C) isolated from tissues versus blood (data from GSE117970). Left, pathways ranked by significance (false-discovery rate q-values), with complement pathways highlighted in red and integrin pathways in yellow. Right, GSEA plots for the top complement pathways in A-C, respectively. (D-F) Expression of C3 mRNA in CD4+ (D) and CD8+ (E) T cells isolated from lung versus spleen (data from GSE94964) and monocytes and macrophages (F) isolated from tissues versus blood (data from GSE117970). (G) Schematic depicting the methicillin-resistant Staphylococcus aureus (MRSA) ear infection model. Wild type (WT) or C3-Td Tomato reporter mice (C3-TdT) were infected in the ear followed by intravital microscopy and blood and organ harvest at day 7 post-infection. (H) Still capture of an intravital imaging movie (see Movie S1) of an MRSA-infected ear section of a WT (left) and C3-TdT reporter mouse (right) at day 7 post-infection. Bar, 30 μm. (I) Representative FACS analysis (top) and cumulative data (below) showing Td Tomato reporter activity in CD4+ (n=7) and CD8+ (n=5) T cells of WT and C3-TdT mice from different tissues at day 7. (J) Representative FACS analysis (left) and cumulative data (right) showing Td Tomato reporter activity in tissue macrophages of WT and C3-TdT mice from the site of infection at day 7 (n=5). I-J show cumulative data from 3–5 experiments. Bars show mean + SEM. * p<0.05, ** p <0.01, ***p < 0.001, ****p < 0.0001. See also Figure S1 and Table S1.

Article Snippet: Human bulk CD4 + or CD8 + T cells were isolated from PBMCs (obtained from freshly drawn blood after centrifugation using Lymphoprep separation medium (Corning, Vienna, VA) using either the MACS Human CD8 + T cell Isolation Kit (130–096-495) from Miltenyi Biotech, (Bergisch Gladbach, Germany), the Negative Selection EasySep CD4 T cell kit (17951) or RosetteSep Human CD4 Cell Isolation Kit (15062) from Stemcell Technologies (Vancouver, Canada) according to the manufacturers’ instructions.

Techniques: Isolation, Expressing, Infection, Intravital Microscopy, Imaging, Activity Assay

Journal: Immunity

Article Title: Diapedesis-induced integrin signalling via LFA-1 facilitates tissue immunity by inducing intrinsic complement C3 expression in immune cells

doi: 10.1016/j.immuni.2020.02.006

Figure Lengend Snippet: (A) Schematic representation of the in vitro transmigration assay utilizing a trans-well system with HUVEC cells and sorted naive and memory CD4+ T cells. (B-D) Transmigration (B) of healthy donor naïve and memory CD4+ T cells across a trans-well system in the presence and absence of IL-1β (induces ICAM-1 expression by HUVEC cells), with and without DEL-1 (a specific inhibitor of LFA-1/ICAM-1 interaction) and expression of C3 (C) and IFNG (D) mRNA by transmigrated cells. Cumulative data from n=6–9 independent experiments. Bars show mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005. See also Figure S3.

Article Snippet: Human bulk CD4 + or CD8 + T cells were isolated from PBMCs (obtained from freshly drawn blood after centrifugation using Lymphoprep separation medium (Corning, Vienna, VA) using either the MACS Human CD8 + T cell Isolation Kit (130–096-495) from Miltenyi Biotech, (Bergisch Gladbach, Germany), the Negative Selection EasySep CD4 T cell kit (17951) or RosetteSep Human CD4 Cell Isolation Kit (15062) from Stemcell Technologies (Vancouver, Canada) according to the manufacturers’ instructions.

Techniques: In Vitro, Transmigration Assay, Expressing

Journal: Immunity

Article Title: Diapedesis-induced integrin signalling via LFA-1 facilitates tissue immunity by inducing intrinsic complement C3 expression in immune cells

doi: 10.1016/j.immuni.2020.02.006

Figure Lengend Snippet: (A) Schematic depicting the integrins and selectins (with respective binding partners in parentheses) involved in rolling, adhesion and transmigration assessed for C3-dependent Th1 induction. (B) Reporter activity of splenic CD4+ T cells from the C3-Td tomato reporter mouse activated with antibodies to CD3 or CD3+CD28 with or without increasing concentrations of immobilized ICAM-1 for seven days and C3-Td Tomato signal measured by flow cytometry. Shown are cumulative data from n=5 independent experiments. (C) C3 mRNA (above) and protein (below) expression over time in healthy donor naive and memory CD4+ T cells cultured in the presence or absence of ICAM-1 alone. Cumulative data (mean ± SEM; n=3 independent experiments); both the C3 mRNA and protein expression curves are higher for both memory and naive cells with ICAM-1 compared to without ICAM-1 (p<0.001 for mRNA expression in both cell types; p<0.0001 for C3 protein in naive T cells and p<0.01 for C3 protein in memory T cells). (D-E) Confocal images for C3 mRNA (using the PrimeFlow assay) and IFN-γ protein (D) and flow cytometry histograms for C3 mRNA (PrimeFlow assay) (E) in bulk CD4+ T cells activated for 36h in the presence and absence of ICAM-1. Shown are representative examples of four independent experiments (n=4). Size bar in ‘D’, 2 μm. Bars show mean + SEM throughout. *p < 0.05, **p < 0.01, ****p < 0.0001. See also Figure S2.

Article Snippet: Human bulk CD4 + or CD8 + T cells were isolated from PBMCs (obtained from freshly drawn blood after centrifugation using Lymphoprep separation medium (Corning, Vienna, VA) using either the MACS Human CD8 + T cell Isolation Kit (130–096-495) from Miltenyi Biotech, (Bergisch Gladbach, Germany), the Negative Selection EasySep CD4 T cell kit (17951) or RosetteSep Human CD4 Cell Isolation Kit (15062) from Stemcell Technologies (Vancouver, Canada) according to the manufacturers’ instructions.

Techniques: Binding Assay, Transmigration Assay, Activity Assay, Flow Cytometry, Expressing, Cell Culture

Journal: Immunity

Article Title: Diapedesis-induced integrin signalling via LFA-1 facilitates tissue immunity by inducing intrinsic complement C3 expression in immune cells

doi: 10.1016/j.immuni.2020.02.006

Figure Lengend Snippet: (A) IFN-γ secretion (left) and intracellular C3a generation (right) by healthy donor CD4+ T cells activated as shown in the presence and absence of ICAM-1 with and without a cell-permeable cathepsin L inhibitor (CTSLi) for 72h (n=4 independent experiments). (B) Representative flow cytometry plots from n=4 independent experiments showing C3 mRNA and the mTOR down-stream target S6 kinase phosphorylated at ser235/ser236 (p-S6) in healthy donor CD4+ T cells activated as shown in the presence or absence of ICAM-1 for 36h. (C) Extracellular acidification rate (ECAR, a measure of glycolysis) and oxygen consumption rate (OCR, a measure of oxidative phosphorylation) in healthy donor naive CD4+ T cells activated as shown in the presence or absence of ICAM-1 with and without a CTSL inhibitor for 36h. Shown are representative ECAR (above) and OCR (middle) graphs together with cumulative data of the maximal respiration and glycolysis (below) from n=3 independent experiments. NA, non-activated; FCCP, p-trifluoromethoxyphenyl hydrazone; OM, oligomycin; ROT, rotenone. Bars show mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. See also Figure S4.

Article Snippet: Human bulk CD4 + or CD8 + T cells were isolated from PBMCs (obtained from freshly drawn blood after centrifugation using Lymphoprep separation medium (Corning, Vienna, VA) using either the MACS Human CD8 + T cell Isolation Kit (130–096-495) from Miltenyi Biotech, (Bergisch Gladbach, Germany), the Negative Selection EasySep CD4 T cell kit (17951) or RosetteSep Human CD4 Cell Isolation Kit (15062) from Stemcell Technologies (Vancouver, Canada) according to the manufacturers’ instructions.

Techniques: Flow Cytometry, Phospho-proteomics

Journal: Immunity

Article Title: Diapedesis-induced integrin signalling via LFA-1 facilitates tissue immunity by inducing intrinsic complement C3 expression in immune cells

doi: 10.1016/j.immuni.2020.02.006

Figure Lengend Snippet: (A-D) C3 mRNA expression in naive (A and B) and memory (C and D) CD4+ T cells activated as indicated in the presence or absence of ICAM-1 with and without a cell-permeable AP-1 inhibitor (AP-1 inh.) or DMSO carrier control at 72h post activation. Shown are representative flow cytometry histograms of (A and C) and cumulative data from n=5 (B) and n=4 (D) independent experiments. NA, non-activated. Error bars represent mean + SEM. **p < 0.01 ***, p < 0.005, ****p < 0.001. See also Figure S5.

Article Snippet: Human bulk CD4 + or CD8 + T cells were isolated from PBMCs (obtained from freshly drawn blood after centrifugation using Lymphoprep separation medium (Corning, Vienna, VA) using either the MACS Human CD8 + T cell Isolation Kit (130–096-495) from Miltenyi Biotech, (Bergisch Gladbach, Germany), the Negative Selection EasySep CD4 T cell kit (17951) or RosetteSep Human CD4 Cell Isolation Kit (15062) from Stemcell Technologies (Vancouver, Canada) according to the manufacturers’ instructions.

Techniques: Expressing, Control, Activation Assay, Flow Cytometry

Journal: Immunity

Article Title: Diapedesis-induced integrin signalling via LFA-1 facilitates tissue immunity by inducing intrinsic complement C3 expression in immune cells

doi: 10.1016/j.immuni.2020.02.006

Figure Lengend Snippet: (A) Steady-state expression of C3 and IFNG mRNA in paired CD4+ T cells drawn from blood and synovial fluid of pediatric patients with juvenile idiopathic oligo-arthritis (n=4; Table S2). Bars show mean + SEM. (B) IFN-γ secretion by paired CD4+ T cells drawn from blood and synovial fluid of two patients with rheumatoid arthritis (RA) activated in vitro with antibodies against CD3 with, or without, ICAM-1. (C-D) C3 mRNA expression by synovial T cells of patients with either oligo-arthritis (OA), non-inflamed RA or inflamed RA derived from an independent dataset (Zhang et al., 2019) (C) and receiver operating characteristic curves showing performance of C3 and IFNG mRNA expression in synovial T cells as biomarkers to distinguish inflamed from un-inflamed RA (D). (E) LFA-1 expression (top), C3 mRNA (middle) and IFN-γ secretion (bottom) by peripheral blood CD4+ T cells from patients with LFA-1 mutations (LAD-1 disease) (see Table S3) and age- and sex-matched controls activated in vitro as shown with, and without ICAM-1 for 72h. Data are from three patients, two of whom donated twice, and five controls. Bars represent mean of duplicate measurements per subject. (F) regression lines showing correlation between IFN-γ and LFA-1 expression (left) and between C3 mRNA and LFA-1 expression (right) from primary data in (E). 95% confidence intervals of the regression line are shown and individual donors are marked. (G) IFN-γ secretion by peripheral blood CD4+ T cells from three patients with LAD-1 disease after transduction with control adenovirus or adenovirus encoding C3a, electroporation with mRNA encoding GFP or C3a or electroporation with BSA or C3H2O protein, as indicated. Cells were activated with anti-CD3+CD46+ICAM-1 after transduction or electroporation. Each patient has been labelled, two of whom donated three times, and the over-expression method shown by color-coding. *p<0.05. See also Figure S6 and Tables S2 and S3.

Article Snippet: Human bulk CD4 + or CD8 + T cells were isolated from PBMCs (obtained from freshly drawn blood after centrifugation using Lymphoprep separation medium (Corning, Vienna, VA) using either the MACS Human CD8 + T cell Isolation Kit (130–096-495) from Miltenyi Biotech, (Bergisch Gladbach, Germany), the Negative Selection EasySep CD4 T cell kit (17951) or RosetteSep Human CD4 Cell Isolation Kit (15062) from Stemcell Technologies (Vancouver, Canada) according to the manufacturers’ instructions.

Techniques: Expressing, In Vitro, Derivative Assay, Transduction, Control, Electroporation, Over Expression

Journal: Immunity

Article Title: Diapedesis-induced integrin signalling via LFA-1 facilitates tissue immunity by inducing intrinsic complement C3 expression in immune cells

doi: 10.1016/j.immuni.2020.02.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human bulk CD4 + or CD8 + T cells were isolated from PBMCs (obtained from freshly drawn blood after centrifugation using Lymphoprep separation medium (Corning, Vienna, VA) using either the MACS Human CD8 + T cell Isolation Kit (130–096-495) from Miltenyi Biotech, (Bergisch Gladbach, Germany), the Negative Selection EasySep CD4 T cell kit (17951) or RosetteSep Human CD4 Cell Isolation Kit (15062) from Stemcell Technologies (Vancouver, Canada) according to the manufacturers’ instructions.

Techniques: Virus, Generated, Control, Isolation, Recombinant, Purification, Staining, Diagnostic Assay, Cell Isolation, Enzyme-linked Immunosorbent Assay, Labeling, Gene Expression, Reverse Transcription, Synthesized, Software, Transfection, Imaging, Flow Cytometry, Microscopy, Western Blot

Journal: Frontiers in Immunology

Article Title: Human Regulatory Dendritic Cells Develop From Monocytes in Response to Signals From Regulatory and Helper T Cells

doi: 10.3389/fimmu.2020.01982

Figure Lengend Snippet: Human monocytes cultured with defined mixtures of Tregs and Th differentiate into cells that induce immune suppressive CD4 + FoxP3 + Tregs. (A) Dotplots and histograms showing CFSE dilution of CD4 + T cells cultured with monocyte-derived cells from the CD4 + T cell-monocyte cultures. (B) Graphical representation of statistical analysis of CFSE – cell percentage (mean ± SEM, n = 3) obtained from (A) . Similar results were obtained in two additional experiments. (C) Proliferation index calculated by Flowjo. (D) Expression of CD25 vs. FoxP3 in CFSE – CD2 + CD4 + cells. Numbers adjacent to gated areas indicate percentage of gated cells. (E,F) Graphical representation of statistical analysis of the MFI of CD25 and the percentage of CD25 high FoxP3 high cells in CD2 + CD4 + CFSE – proliferated T cells. (G) CD14 + monocytes were cultured with Tregs and Th at a ratio of 10:1:1 in the presence of anti-IL-10, 2 μg/ml; anti-TGFβ, 2 μg/ml or isotype control antibody, 5 μg/ml. After 72 h, 1 μg/ml LPS was added, and 16 h later FACS-purified myeloid cells (DR + CD2 – ) were further cultured with MACS sorted CFSE-labeled naïve allogeneic CD4 + T cells. The percentage of FoxP3 high cells in CD2 + CD4 + CFSE – cells was determined 6 days later. Mean ± SEM, n = 5. Similar results were obtained in two additional experiments. * P < 0.05; ** P < 0.001; *** P < 0.0001; ns, not significant. Data were analyzed with one-way ANOVA, followed by Dunnett’s test for multiple comparisons. (H,I) 10 5 CFSE labeled CD45RA + CD4 + T cells were cultured with 5 × 10 4 allogeneic myeloid cells purified from 3-day cultures of monocytes, Tregs and Th at 10:1:1. After 6 days the cells were analyzed by flow cytometry. Responder CD4 + T cells were gated as CD2 + CD4 + . (H) CFSE – FoxP3 + DC Reg -induced Tregs were analyzed for CD25 expression. (I) CD25 expression was used as the basis for sorting CD2 + CD4 + CFSE – cells. Percentages of FoxP3 – and FoxP3 + cells in CD2 + CD4 + CFSE – CD25 + cells are shown. (J–M) CD2 + CD4 + CFSE – CD25 + cells induced by 1:1 (Th:Treg), Th or Treg-derived myeloid cells were purified by FACS and cultured in a new MLR containing 10 5 CFSE labeled allogeneic CD45RA + CD4 + cells and irradiated DCs (5 × 10 4 ) and anti-CD3 (0.5 ng/ml, plate-bound). After 84–90 h, proliferation of CFSE-labeled naïve responder CD4 + T cells was analyzed by flow cytometry. Cells were gated as DAPI – CD2 + CD4 + . Data are representative of 2 independent experiments. (J–L) Dose-dependent suppression of induced Tregs on responder CD4 + T cells. (L) Heatmap of statistical analysis of the responder CD4 + T cell proliferation as indicated by CFSE in each division defined by Flowjo. Mean ± SEM, n = 3. Similar results were obtained in both experiments.

Article Snippet: The CD4 + T cells for these assays were purified from PBMCs using a RosetteSep CD4 + Human T cell Isolation Kit (Stem Cell Technologies) followed by magnetic purification (>95% CD2 + CD4 + CD45RA + cells by flow cytometry) using a Naïve CD4 + T Cell Isolation Kit II (Miltenyi Biotec) and subsequently labeled with CFSE.

Techniques: Cell Culture, Derivative Assay, Expressing, Control, Purification, Labeling, Flow Cytometry, Irradiation

Journal: Frontiers in Immunology

Article Title: Human Regulatory Dendritic Cells Develop From Monocytes in Response to Signals From Regulatory and Helper T Cells

doi: 10.3389/fimmu.2020.01982

Figure Lengend Snippet: Blockade of CTLA-4, IL-10 and TGF-β prevents DC Reg differentiation. (A–D) CD14 + monocytes were cultured for 4 days with Tregs or Th alone, or with Tregs and together at a 1:1 ratio in the presence of anti-CTLA-4 (5 μg/ml), anti-IL-10 (2 μg/ml), anti-TGFβ (2 μg/ml), or a mixture of these antibodies or isotype control antibody (5 μg/ml). (A) Images were captured with a bright-field DIC microscope on day 4. Original magnification 200×. (B) MFIs of DC-associated molecules on monocyte-derived cells are shown after excluding CD2 + HLA-DR – T cells. (C) On day 4, moDCs (HLA-DR + CD2 – cells) from the cultures in (A,B) were purified by FACS, and 200,000 cells were cultured in 200 μl containing 1 μg/ml LPS. After 16h, IL-10, IL-1β, IL-6 and TNFα levels in the cell-free supernatants were determined by Luminex. (D) On day 3, 1 μg/ml LPS was added to selected wells, and 16h later FACS-purified DC subsets were further cultured with MACS sorted CFSE-labeled naïve allogeneic CD4 + T cells. After another 6 days, CD2 + CD4 + T cells were analyzed by flow cytometry. Mean ± SEM of 4 donors. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant. Data were analyzed with one-way ANOVA, followed by Dunnett’s test for multiple comparisons.

Article Snippet: The CD4 + T cells for these assays were purified from PBMCs using a RosetteSep CD4 + Human T cell Isolation Kit (Stem Cell Technologies) followed by magnetic purification (>95% CD2 + CD4 + CD45RA + cells by flow cytometry) using a Naïve CD4 + T Cell Isolation Kit II (Miltenyi Biotec) and subsequently labeled with CFSE.

Techniques: Cell Culture, Control, Microscopy, Derivative Assay, Purification, Luminex, Labeling, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Human Regulatory Dendritic Cells Develop From Monocytes in Response to Signals From Regulatory and Helper T Cells

doi: 10.3389/fimmu.2020.01982

Figure Lengend Snippet: CRC CD4 + T cells induce DC Reg formation. (A) Left: representative human colon cancer tissue stained with antibodies against FoxP3 (brown) and CD14 (blue). Black arrows represent potential monocyte-Treg interactions. Right: representative human colon cancer tissue stained with antibodies against FoxP3 (brown) and T-bet (blue). Magnification 400×. (B) Representative flow cytometry analysis of CD25 + CD127 low Tregs in CD45 + CD2 + CD4 + T cells from colon cancer sample. (C) Comparison of the CD25 + CD127 low Treg percentages from healthy donor peripheral blood and colon cancer samples. Mean ± SEM. **** P < 0.0001, unpaired T test. (D,E) Lin – CD45 + CD2 + CD4 + T cells were purified from human CRC and co-cultured with peripheral blood CD14 + monocytes at a 1:10 ratio in the presence of anti-CD3 mAb for 4 days. (D) Images were captured with a bright-field DIC microscope on day 4. Original magnification 200×. (E) Flow cytometry analysis of the resultant antigen presenting cells (HLA-DR + CD2 – ) (red line) compared to monocytes cultured alone (gray line) or with GM-CSF and IL-4 (black line). (F) CD11c + HLA – DR + CD2 – cells were purified via FACS and stimulated for 16 h with LPS. Cells were washed extensively and cultured with CFSE-labeled allogeneic naïve CD4 + T cells in a MLR. The percentage of FoxP3 + cells in CD2 + CD4 + CFSE – cells was determined 6 days later (red line). Naïve CD4 + T cells cultured alone (black line) are shown as controls. Shaded histograms represent isotype controls. Data are representative of 3 independent experiments. (G) In the presence of anti-CD3 mAb, CD14 + monocytes were cultured with bulk CD45 + CD2 + CD4 + T cells sorted from CRC, in the presence of a mixture of anti-CTLA-4 (5 μg/ml), anti-IL-10 (2 μg/ml), anti-TGFβ (2 μg/ml), or isotype control antibody (5 μg/ml). At day 4, cells in culture were analyzed by flow cytometry for the indicated surface markers. (H) CD14 + monocytes were cultured with bulk CD45 + CD2 + CD4 + T cells sorted from CRC (red) or healthy donor peripheral blood (black). CD25 + CD127 low Treg percentages were analyzed before coculture. The graph shows the correlation between the percentage of Tregs in CD2 + CD4 + T cells prior to their initial culture with monocytes ( X axis) and the percentage of CFSE – FoxP3 + T cells among the responder CD4 + T cells cultured with the DCs generated in the initial culture ( Y axis). Linear regression was determined by prism. R 2 = 0.9509. P = 0.0002. (I) Representative flow cytometry analysis of human CRC DCs. DAPI – CD45 + cells were further gated with CD11c, HLA-DR. (J) Cytospin of freshly FACS-sorted CD11c + HLA-DR + DCs with Grunwald-Giemsa staining from CRC. Scale bar as indicated. (K) Flow cytometry analysis of CD40, CD80, CD86 and CD274 expression on gated CD11c + HLA-DR + DCs. Red line: Ab staining. Shaded histograms represent isotype controls. (L) CD45 + CD11c + HLA – DR + DCs from CRC were sorted by FACS and cultured with CFSE-labeled naïve allogeneic CD4 + T cells. The percentage of FoxP3 + cells in CD2 + CD4 + CFSE – cells was determined 6 days later.

Article Snippet: The CD4 + T cells for these assays were purified from PBMCs using a RosetteSep CD4 + Human T cell Isolation Kit (Stem Cell Technologies) followed by magnetic purification (>95% CD2 + CD4 + CD45RA + cells by flow cytometry) using a Naïve CD4 + T Cell Isolation Kit II (Miltenyi Biotec) and subsequently labeled with CFSE.

Techniques: Staining, Flow Cytometry, Comparison, Purification, Cell Culture, Microscopy, Labeling, Control, Generated, Expressing