Journal: Nature immunology

Article Title: Autocrine Vitamin D-signaling switches off pro-inflammatory programs of Th1 cells

doi: 10.1038/s41590-021-01080-3

Figure Lengend Snippet: a-b , UMAP representation of scRNAseq showing main clusters of cells from bronchoalveolar lavage fluid (BALF) of patients with COVID-19 and healthy controls ( a ) and dot plot depicting expression of select marker genes for each cluster ( b ). Highlighted in both a and b are clusters 2 and 12, which represent T lymphocytes. c , Dot plot showing expression of select marker genes for clusters of cells depicted in . d , UMAP projection of scRNAseq showing sub-clustering of T cells from bronchoalveolar lavage fluid (BALF) of healthy controls (above) and patients with COVID-19 (below). e , GSEA showing genes more highly expressed in bulk RNA-seq of BALF cells obtained from patients ( n =8) with COVID-19 compared to healthy controls ( n =20) are enriched in Th1 genes. Box and whisker plots (right) shows the expression of IL10 mRNA in these samples, indicating the median and extending to the minimum, maximum, 25% and 75% quartiles. Data in a-b are from n =9 patients with COVID-19 and n =4 healthy controls, sourced from GSE145926 and GSE122960. Data in c-d are from the same sources but with n =8 patients with COVID-19 and n =3 healthy controls (one sample from each was removed due to too low CD4 + T cell numbers). Data in e are from n =8 patients with COVID-19 and n =20 healthy subjects, obtained from HRA000143. **** p<0.0001 by two-sided Mann-Whitney U-test.

Article Snippet: CD4 + T cells were enriched using RosetteSep Human CD4 + enrichment cocktail (Stem Cell) with CD4 + CD25 − cells obtained by depletion of CD25 + T cells using CD25 positive selection (CD25 microbeads II, Human, Miltenyi Biotec) followed by unlabeled cell collection.

Techniques: Expressing, Marker, RNA Sequencing, Whisker Assay, MANN-WHITNEY

Journal: Nature immunology

Article Title: Autocrine Vitamin D-signaling switches off pro-inflammatory programs of Th1 cells

doi: 10.1038/s41590-021-01080-3

Figure Lengend Snippet: a-b , UMAP representation of scRNAseq showing main clusters of cells from peripheral blood mononuclear cells (PBMC) of patients with COVID-19 and healthy controls ( a ) and dot plot depicting expression of select marker genes for each cluster ( b ). Highlighted in both a and b are clusters 4, 5 and 6, which represent CD4 + T lymphocytes. c , Violin plots showing expressions of Th1, Th2 and Th17 genes, respectively, summarized as module scores, in PBMC CD4 + T cells of patients with COVID-19 and healthy controls. Data in a-c are from n =6 patients with COVID-19 and n =6 healthy subjects, obtained from GSE150728.

Article Snippet: CD4 + T cells were enriched using RosetteSep Human CD4 + enrichment cocktail (Stem Cell) with CD4 + CD25 − cells obtained by depletion of CD25 + T cells using CD25 positive selection (CD25 microbeads II, Human, Miltenyi Biotec) followed by unlabeled cell collection.

Techniques: Expressing, Marker

Journal: Nature immunology

Article Title: Autocrine Vitamin D-signaling switches off pro-inflammatory programs of Th1 cells

doi: 10.1038/s41590-021-01080-3

Figure Lengend Snippet: a , Representative flow cytometry showing IFN-γ and IL-10 in CD4 + T (Th) cells activated with α-CD3+α-CD46 and the four quadrants (A, B, C and D) from which cells were flow-sorted for transcriptome analysis. Live and single cells are pre-gated. b , Venn diagram showing number of DEGs (≥±1.5-fold at unadjusted p-value<0.05 using ANOVA) comparing cells in quadrants B, C and D against A, respectively ( n =4 experiments). c , Enrichment of gene ontology molecular function terms in shared DEGs (intersect of Venn diagram in b ), ranked by statistical significance. Marked are terms corresponding to transcription factor (TF) activity. d , Heatmap of induced TFs in α-CD3+α-CD46-activated Th cells at each stage of the life-cycle shown in a . Highlighted are VDR and expression of CYP27B1 . e , EnrichR-predicted ENCODE and ChEA (ChIP enrichment analysis) TFs regulating the DEGs between COVID-19 vs. healthy donor Th cells (upper panel) and lung biopsies (lower panel). Shown are Benjamini-Hochberg adjusted p-values from hypergeometric tests. f , VDR (left panel) and CYP27B1 (right panel) mRNA in Th cells activated, or not, as indicated, with or without cathepsin L inhibitor (CTSL inh.) ( n =5 experiments). g , VDR (left panel) and CYP27B1 (right panel) mRNA in Th cells of a patient with CD46-deficiency, activated, or not, as indicated ( n =3 experiments). h-i , Representative flow cytometry ( h ) and cumulative data from n =6 independent experiments ( i ) showing IFN-γ and IL-10 in Th cells activated with α-CD3+α-CD46 with, or without, carrier, active [1,25(OH)2D3] or inactive [25(OH)D3] VitD. j , IL10 in Th cells activated with α-CD3+α-CD46 with, or without, inactive [25(OH)D3] VitD, with siRNA targeting VDR , CTSL or CYP27B1 , or non-targeting siRNA (NT) ( n =5 experiments). Data in a-d are from GSE119416. Data in e , upper panel are from GSE145926 and GSE122960. Data in e , lower panel are from GSE147507. Data in g are from microarrays in . Bars in f-g and i show mean + sem; box plots in j show median value and the range extends from minimum to maximum. All statistical tests are two-sided. *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001 by ANOVA.

Article Snippet: CD4 + T cells were enriched using RosetteSep Human CD4 + enrichment cocktail (Stem Cell) with CD4 + CD25 − cells obtained by depletion of CD25 + T cells using CD25 positive selection (CD25 microbeads II, Human, Miltenyi Biotec) followed by unlabeled cell collection.

Techniques: Flow Cytometry, Activity Assay, Expressing

Journal: Nature immunology

Article Title: Autocrine Vitamin D-signaling switches off pro-inflammatory programs of Th1 cells

doi: 10.1038/s41590-021-01080-3

Figure Lengend Snippet: CD4 + T cells were activated as before using anti-CD3 + anti-CD46 in culture plates. After 48h cells were stained for IFN-γ and IL-10 and co-stained with CD25 or CD69. Separately, CTV-labelled CD4 + T cells were activated in the same manner. a-b , representative flow cytometry histograms ( a ) and cumulative data from n =3 independent experiments ( b ) of CD25 and CD69 expression in cells at each stage of cytokine secretion. c-d , representative CTV dilution representing cells that had undergone proliferation ( c ) and cumulative data from n =3 independent experiments ( d ). Box and whisker plots in b and d show the medians and extend to the minimum, maximum, 25% and 75% quartiles. * p<0.05 by two-sided ANOVA. All other comparisons were non-significant.

Article Snippet: CD4 + T cells were enriched using RosetteSep Human CD4 + enrichment cocktail (Stem Cell) with CD4 + CD25 − cells obtained by depletion of CD25 + T cells using CD25 positive selection (CD25 microbeads II, Human, Miltenyi Biotec) followed by unlabeled cell collection.

Techniques: Staining, Flow Cytometry, Expressing, Whisker Assay

Journal: Nature immunology

Article Title: Autocrine Vitamin D-signaling switches off pro-inflammatory programs of Th1 cells

doi: 10.1038/s41590-021-01080-3

Figure Lengend Snippet: a-c , Volcano plots showing differentially expressed genes (DEGs) following activation of CD4 + T cells with α-CD3+α-CD46, comparing IFN-γ + IL-10 − cells ( a ), IFN-γ + IL-10 + cells ( b ) and IFN-γ − IL-10 + cells ( c ) to IFN-γ − IL10 − cells, respectively. DEGs are defined as at least 1.5-fold change in either direction at unadjusted p-value <0.05 using two-sided ANOVA. Marked in a-c are the IFNG , IL10 , VDR and CYP27B1 genes. d , Heatmap showing expression of the 2023 shared DEGs in a-c . e , ClueGo analysis for molecular function terms in the 2023 DEGs shown in d represented as a Cytoscape visualization. Genes are shown in grey, enriched molecular function terms are in red scaled to reflect fold enrichment and edges link genes to molecular function terms. Node sizes reflect enrichment significance. Related terms grouped as families within yellow circles. Four such families represent transcriptional regulation of gene expression and are shown in the inset on the right. f , The top 14 transcription factor molecular function terms are shown, with associated fold enrichments and FDR q-values. Data in a-f are from n =4 experiments.

Article Snippet: CD4 + T cells were enriched using RosetteSep Human CD4 + enrichment cocktail (Stem Cell) with CD4 + CD25 − cells obtained by depletion of CD25 + T cells using CD25 positive selection (CD25 microbeads II, Human, Miltenyi Biotec) followed by unlabeled cell collection.

Techniques: Activation Assay, Expressing, Gene Expression

Journal: Nature immunology

Article Title: Autocrine Vitamin D-signaling switches off pro-inflammatory programs of Th1 cells

doi: 10.1038/s41590-021-01080-3

Figure Lengend Snippet: a , Western blot (left) and cumulative data (right) for VDR at days 1, day 3 and day 5, with Hsp90 as loading control, in both carrier and VitD treated CD4 + T cells. b , Representative immunoblots for VDR and indicated housekeeping proteins in nuclear and cytoplasmic extracts of carrier and VitD treated CD4 + T cells. c , Co-localization of VDR and DAPI in carrier and VitD treated CD4 + T cells, measured on day 2 using ImageStream. Shown are representative frequency histograms indicating overlap between VDR and DAPI in the entire population (left), and cumulative data from n =3 independent experiments (right). d , Cell death assessed by live/dead stain and proliferation assessed by CFSE dilution in CD4 + T cells treated with carrier or VitD after 3 days of culture. e , GSEA showing genes more highly expressed in CD4 + T cells treated with carrier compared to VitD are enriched in Tr1-induced genes (left panel) and genes more highly expressed in CD4 + T cells treated with VitD compared to carrier are enriched in Tr1-repressed genes (right panel; curated from GSE139990). f , Representative flow cytometry plot showing CD49b and LAG-3 expression in CD4 + T cells, with carrier and VitD treatment (left), and quantification of cumulative data (right). g , Top 5 MSigDB canonical pathways enriched in DEGs of VitD vs carrier treated CD4 + T cells (see ). Unless indicated, all cells in Fig. S6 have been activated with α-CD3+α-CD28. Bars represent mean + sem throughout. All experiments have been carried out n =3 times. Shown in e are unadjusted empirical p-values; NES = normalized enrichment score. *p<0.05, ****p<0.0001 by 2-way ANOVA ( a , f ) and paired t-test ( c ). All statistical analyses are two-sided.

Article Snippet: CD4 + T cells were enriched using RosetteSep Human CD4 + enrichment cocktail (Stem Cell) with CD4 + CD25 − cells obtained by depletion of CD25 + T cells using CD25 positive selection (CD25 microbeads II, Human, Miltenyi Biotec) followed by unlabeled cell collection.

Techniques: Western Blot, Control, Staining, Flow Cytometry, Expressing

Journal: Nature immunology

Article Title: Autocrine Vitamin D-signaling switches off pro-inflammatory programs of Th1 cells

doi: 10.1038/s41590-021-01080-3

Figure Lengend Snippet: a , Differentially expressed genes (DEGs) between VitD and carrier treated CD4 + T cells (see ) ranked by fold change. Each DEG is marked by a blue dot; each differentially expressed cytokine is marked by an orange dot. Select cytokines have been labelled. b , Cytokine concentrations in supernatants of CD4 + T cell cultures after 5 days of treatment with carrier or VitD. c , Heatmap showing mRNA expressions (log2 TPM) of the 25-hydroxylase enzymes (CYP2R1 and CYP27A1), the 1α-hydroxylase enzyme (CYP27B1) and the 24-hydroxylase enzyme (CYP24A1), responsible for the two steps of Vitamin D activation and its subsequent inactivation, respectively. Data are from CD4 + T cells activated with α-CD3+α-CD28 and cultured with either carrier or VitD, or left unactivated. d , Concentrations of indicated cytokines in culture supernatants of CD4 + T cells treated with escalating doses of 25(OH)VitD for 72h. e , IL6 mRNA, fold change compared to day 1 carrier (above), and IL-6 protein concentration in matched supernatants (below), at days 1, 3 and 5 in carrier and VitD-treated CD4 + T cell cultures. f , Representative flow cytometry plot (left) and cumulative data (right) of intracellular IL-6 expression in T cells treated with carrier or VitD (assay carried out on day 3). Cells gated based on lymphocyte gate (forward scatter, side scatter), singlets, live cells and CD4 + cells. g , IL-10 concentrations in supernatants of CD4 + T cells cultured in the presence of increasing concentrations of IL-6 for 72 hours. h , IL-17 concentrations in supernatants of CD4 + T cells cultured in the presence of increasing concentrations of IL-6, with and without VitD for 72 hours. i , Volcano plot representing changes in protein phosphorylation on phospho-kinase array comparing VitD-treated versus carrier treated cells. Data are from n =2 independent experiments. Thresholds for significance have been set at 1.2 fold change in phosphorylation in either direction at p-value <0.05. Please also see . Marked are phosphoproteins that show significant changes in phosphorylation on VitD treatment. j , Quantification of pY-STAT3, STAT3, p-c-JUN, c-JUN and Hsp90 from immunoblots of lysates of CD4 + T cells treated with carrier or VitD with, and without, Tocilizumab (Toc) at the concentrations shown. Bars show mean + sem from n =3 independent experiments. Please also see . k-l , Shown are representative Western blots ( k ) and quantifications ( l ) of STAT3 and HSP90 in Carrier- and VitD-treated CD4 + T cells in the presence and absence of STAT3 siRNA. Data are representative of n =2 experiments carried out. Unless indicated, all cells in Fig. S7 have been activated with α-CD3+α-CD28. Cumulative data in b , d-h , j depict mean + sem. Unless indicated, all experiments have been carried out n =3 times. All statistical tests are two-sided. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by one-way ANOVA ( d , j ), two-way ANOVA ( b , e , h ) and paired t-test ( f ). Statistical comparisons in d and h compare VitD-treated ( d ) or IL-6-treated ( h ) cells against untreated cells.

Article Snippet: CD4 + T cells were enriched using RosetteSep Human CD4 + enrichment cocktail (Stem Cell) with CD4 + CD25 − cells obtained by depletion of CD25 + T cells using CD25 positive selection (CD25 microbeads II, Human, Miltenyi Biotec) followed by unlabeled cell collection.

Techniques: Activation Assay, Cell Culture, Protein Concentration, Flow Cytometry, Expressing, Phospho-proteomics, Western Blot

Journal: Nature immunology

Article Title: Autocrine Vitamin D-signaling switches off pro-inflammatory programs of Th1 cells

doi: 10.1038/s41590-021-01080-3

Figure Lengend Snippet: a , histograms showing average cuts per bp in relation to the summits of c-JUN, STAT3, VDR and BACH2 peaks in VitD-treated CD4 + T cells. Shown are data from CUT&RUN (c-JUN) and CUT&Tag (VDR, STAT3 and BACH2) carried out with IgG (cyan) or antibodies specific to each TF (dark blue). p-values from K-S tests are indicated. b , heatmaps showing H3K27Ac and c-JUN signals at VitD-repressed and VitD-induced peaks over time. Time points are indicated. c-d , genome browser tracks at the CTLA4 ( c ) and STAT3 ( d ) loci showing H3K27Ac, c-JUN, BACH2, STAT3 and VDR binding in Carrier and VitD-treated cells. Red and blue dots represent peaks in Carrier and VitD-treated cells, respectively. SE denotes super-enhancer regions. Track heights are indicated on the left corner for each track. All cells in Fig. S8 have been activated with α-CD3+α-CD28.

Article Snippet: CD4 + T cells were enriched using RosetteSep Human CD4 + enrichment cocktail (Stem Cell) with CD4 + CD25 − cells obtained by depletion of CD25 + T cells using CD25 positive selection (CD25 microbeads II, Human, Miltenyi Biotec) followed by unlabeled cell collection.

Techniques: Binding Assay

Journal: Nature immunology

Article Title: Autocrine Vitamin D-signaling switches off pro-inflammatory programs of Th1 cells

doi: 10.1038/s41590-021-01080-3

Figure Lengend Snippet: a-c , GSEA comparing the transcriptomes of carrier and VitD-treated CD4 + T cells against BACH2-bound BACH2-induced genes ( a ) and -repressed genes ( b ). Shown in c are the leading edges of the two GSEA enrichment plots in a-b . Marked in a and c is the IL-6 receptor (IL6R). d , GSEA comparing enrichment in VitD-repressed genes of the transcriptomes of VitD-treated BACH2 WT/WT haplo-sufficient and BACH2 WT/L24P haplo-insufficient CD4 + T cells. e , Circos diagram showing VitD-induced and repressed genes in VitD-treated BACH2 WT/WT haplo-sufficient and BACH2 WT/L24P haplo-insufficient CD4 + T cells. Cords join shared genes in patient and control. Indicated are the shared VitD-induced genes ( CYP24A1 , CD38 and IL6 ) and genes only induced by VitD in the presence of two wild-type copies of BACH2 ( IL10 and IL6R ). f , Genome browser tracks showing Bach2 ChIP-seq at the IL6ra locus and expression of IL6ra mRNA in CD4 + T cells of Bach2 wild-type ( Bach2 +/+ ) and knock-out ( Bach2 -/- ) mice. Track heights are indicated on the left corner for each track. Source data are from GSE45975. Empirical p-values are shown in a-b and d . NES = normalized enrichment score.

Article Snippet: CD4 + T cells were enriched using RosetteSep Human CD4 + enrichment cocktail (Stem Cell) with CD4 + CD25 − cells obtained by depletion of CD25 + T cells using CD25 positive selection (CD25 microbeads II, Human, Miltenyi Biotec) followed by unlabeled cell collection.

Techniques: Control, ChIP-sequencing, Expressing, Knock-Out

Journal: Nature immunology

Article Title: Autocrine Vitamin D-signaling switches off pro-inflammatory programs of Th1 cells

doi: 10.1038/s41590-021-01080-3

Figure Lengend Snippet: a , Violin plots showing expressions of VitD-repressed genes, summarized as module scores, in BALF Th cells of patients with COVID-19 and healthy controls. Exact p-values in a have been calculated using two-tailed Wilcoxon tests. b , GSEA showing enrichment in VitD-repressed genes within genes more highly expressed in scRNAseq CD4 + BALF T cells of patients with COVID-19 compared to healthy controls. c , Correlation between module scores of Th1-genes and VitD-repressed genes on a per cell basis in BALF Th cells of patients with COVID-19 and healthy controls. Pearson r and exact p-values are shown. d , Receiver operating characteristic (ROC) curve, evaluating the performance of the Th1 and VitD-repressed module scores to distinguish BALF Th cells of patients with COVID-19 from healthy controls. Shown are the area under the curve (AUC) statistics and p-values. e , Analyses showing the performance of all MSigDB canonical and hallmark genesets to distinguish BALF Th cells of patients with COVID-19 from healthy controls, ranked by AUC values. Marked are the top 2 performing genesets in red and the position of the VitD-repressed geneset within the top 1% of all genesets. f , Top 10 drugs predicted (out of 461 significant drugs) to counteract genes induced in BALF Th cells of COVID-19 patients compared to healthy controls, ordered by adjusted p-value. Highlighted in red is alfacalcidol, an FDA-approved active form of VitD. g-h , GSEA showing enrichment in VitD-repressed genes for genes more highly expressed in bulk RNA-seq lung biopsy specimens ( g ) and bulk RNA-seq BALF cells ( h ) of COVID-19 compared to healthy controls. Empirical p-values are shown for GSEA in b , g-h ; NES = normalized expression value. p-values in d are from the Mann-Whitney U-statistic. Data in a-f are from n =8 patients with COVID-19 and n =3 healthy controls, sourced from GSE145926 and GSE122960. Data in g-h are from GSE147507 and HRA000143, respectively, and n numbers are indicated.

Article Snippet: CD4 + T cells were enriched using RosetteSep Human CD4 + enrichment cocktail (Stem Cell) with CD4 + CD25 − cells obtained by depletion of CD25 + T cells using CD25 positive selection (CD25 microbeads II, Human, Miltenyi Biotec) followed by unlabeled cell collection.

Techniques: Two Tailed Test, RNA Sequencing, Expressing, MANN-WHITNEY

Journal: Nature immunology

Article Title: Autocrine Vitamin D-signaling switches off pro-inflammatory programs of Th1 cells

doi: 10.1038/s41590-021-01080-3

Figure Lengend Snippet: a , Violin plots showing expressions of VitD-induced genes, summarized as module scores, of BALF CD4 + T cells of patients with COVID-19 and healthy controls. Data are from n =8 patients with COVID-19 and n =3 healthy controls, sourced from GSE145926 and GSE122960. b , Violin plots showing expressions of VitD-induced and VitD-repressed genes, summarized as module scores, of PBMC CD4 + T cells of patients with COVID-19 and healthy controls. Data are from n =6 patients with COVID-19 and n =6 healthy subjects, obtained from GSE150728. c , Venn diagram showing overlap between COVID-induced genes in CD4 + BALF T cells that are predicted to be normalized by VitD treatment versus those that are predicted to be normalized by steroid drugs. Please also see . d , Schematic model of autocrine VitD-driven Th1 contraction program and potential intervention of impaired COVID-19 program with VitD and cortico-steroids. e , representative flow sorting strategy and example of post-sort purity obtained for isolation of memory CD4 + T cells in this paper.

Article Snippet: CD4 + T cells were enriched using RosetteSep Human CD4 + enrichment cocktail (Stem Cell) with CD4 + CD25 − cells obtained by depletion of CD25 + T cells using CD25 positive selection (CD25 microbeads II, Human, Miltenyi Biotec) followed by unlabeled cell collection.

Techniques: Isolation

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: PLoS Pathogens

Article Title: HIV infects astrocytes in vivo and egresses from the brain to the periphery

doi: 10.1371/journal.ppat.1008381

Figure Lengend Snippet: Neonate NSG mice were xenotransplanted with HIV- or HIV+ NHAs as described in and HIV detection outside of the brain was evaluated by Real-time PCR products (HIV DNA, A , or HIV RNA, B ) from brain, cervical lymph node, spleen, splenocytes outgrowth assay and splenocytes outgrowth assay supernatant added to fresh PBMCs analyzed by electrophoresis and human GAPDH. Each column indicates individual animal. HIV- animals are shown to left of ladder, HIV+ animals shown to right for all gels shown. No template control (NTC) is the negative control. M/F indicates sex and number is animal number from that group. ( C ) GFP expression in splenocytes at day 14 day of culturing the cells and ( D ) flow cytometry of cultured splenocytes stained for CD3+/CD4+/GFP; dot blots (left) and cumulative data on right ( p = 0.05, Mann-Whitney U-test). ( E ) Representative image of supernatant from splenocytes cultured for 14 days from neonates from HIV- animal (top) or HIV+ animal (bottom) on fresh PBMCs stimulated with soluble α-CD3 and α-CD28 IL-2. Egress for neonates was analyzed in n = 7 (HIV-) and 12 (HIV+) mice; n = 3 (HIV-) and 4 (HIV+) representative mice are shown here; n = 3 (HIV-) and 5 (HIV+) representative mice are shown here.

Article Snippet: Monocytes were depleted using RosetteSep Human Monocyte Depletion Cocktail and CD4+ T cells by RosetteSep Human CD4+ Depletion Cocktail (STEMCELL Technologies, Cambridge, MA) and were injected IP with 2 × 10 7 of the remaining PBMCs.

Techniques: Real-time Polymerase Chain Reaction, Electrophoresis, Control, Negative Control, Expressing, Flow Cytometry, Cell Culture, Staining, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: HIV infects astrocytes in vivo and egresses from the brain to the periphery

doi: 10.1371/journal.ppat.1008381

Figure Lengend Snippet: Adult NSG mice were xenotransplanted with HIV- or HIV+ NHAs as described in and HIV detection outside of the brain was evaluated by Real-time PCR products (HIV DNA, A , or HIV RNA, B ) from brain, cervical lymph node, spleen, peripheral lymph node, splenocytes outgrowth assay and splenocytes outgrowth assay supernatant added to fresh PBMCs analyzed by electrophoresis and human GAPDH. Each column indicates individual animal. HIV- animals are shown to left of ladder, HIV+ animals shown to right for all gels shown. No template control (NTC) is the negative control. ( C ) GFP expression in splenocytes at day 14 day of culturing the cells and ( D ) flow cytometry of cultured splenocytes stained for CD3+/CD4+/GFP; dot blots (left) and cumulative data on right ( p = 0.04, Mann-Whitney U-test). ( E ) Representative image of supernatant from splenocytes cultured for 14 days from neonates from HIV- animal (top) or HIV+ animal (bottom) on fresh PBMCs stimulated with soluble α-CD3 and α-CD28 IL-2. Egress was analyzed in n = 7 (HIV-) and 9 (HIV+) mice; n = 3 (HIV-) and 5 (HIV+) representative mice are shown here.

Article Snippet: Monocytes were depleted using RosetteSep Human Monocyte Depletion Cocktail and CD4+ T cells by RosetteSep Human CD4+ Depletion Cocktail (STEMCELL Technologies, Cambridge, MA) and were injected IP with 2 × 10 7 of the remaining PBMCs.

Techniques: Real-time Polymerase Chain Reaction, Electrophoresis, Control, Negative Control, Expressing, Flow Cytometry, Cell Culture, Staining, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: HIV infects astrocytes in vivo and egresses from the brain to the periphery

doi: 10.1371/journal.ppat.1008381

Figure Lengend Snippet: ( A ) Representative image of neonate hippocampus (HPC) immunostained for human T cells (CD3+, magenta), human astrocytes (huGFAP; red), HIV (green) and Nuclei (DAPI, blue) at week 10 post-xenotransplantation and week 4 reconstitution with huPBMCs. Arrows indicates detection of CD3+/HIV+ cell. Scale bar, 10μm. ( B ) Total PBMCs (HIV+) or monocyte/CD4+ T cell depleted PBMCs analyzed by flow cytometry for CD3+/CD4+ positive T cells (top) or CD14 positive or CD14/CD16 positive monocytes before injection into the adult mouse xenotransplanted with HIV VSVg + NHAs. HIV DNA ( C ) and RNA ( D ) from spleen from animals xenotransplanted with HIV VSVg + NHAs and reconstituted with total PBMCs (HIV+) or PBMCs depleted of monocytes and CD4+ T cells (Mono/CD4). No analysis was performed on DNA as Mono/CD4 group had undetectable HIV DNA. RNA is depicted on the right ( p = 0 . 08 , Mann-Whitney U-test). n = 5–7 per group.

Article Snippet: Monocytes were depleted using RosetteSep Human Monocyte Depletion Cocktail and CD4+ T cells by RosetteSep Human CD4+ Depletion Cocktail (STEMCELL Technologies, Cambridge, MA) and were injected IP with 2 × 10 7 of the remaining PBMCs.

Techniques: Flow Cytometry, Injection, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: HIV infects astrocytes in vivo and egresses from the brain to the periphery

doi: 10.1371/journal.ppat.1008381

Figure Lengend Snippet: Human post mortem analysis of HIV+ astrocytes. Post mortem analysis from cortical or hippocampal brain regions of HIV- or HIV+ individuals. Laser capture microdissection (LCM) of GFAP+ cells analyzed for Alu-gag HIV by PCR. RNAscope and staining determined GFAP+ co-localization with gag mRNA, nef DNA and p24. Staining for each patient included analysis of 12 different sections with 20–24 fields per section (~240 fields) from an average tissue size of 1.5±0.75x0.91±0.66 cm. M: male; F: Female; N.A.: not applicable; U.D.: undetectable. Anti-retrovirals: ATV, atazanavir (Reyataz); EFV, efavirenz (Sustiva); RTV, ritonavir (Norvir); TFV, PMPA; tenofivir DF (Viread); 3TC, lamivudine (Epivir); D4T, stavudine (Zerit FTV, SQV2 or FTV saquinavir-sgc (Fortovase);SQV, saquinavir (Invirase); DDI, didanosine (Videx); NFV, nelfinavir (Viracept). Representative staining images of data are shown in Fig 9 .

Article Snippet: Monocytes were depleted using RosetteSep Human Monocyte Depletion Cocktail and CD4+ T cells by RosetteSep Human CD4+ Depletion Cocktail (STEMCELL Technologies, Cambridge, MA) and were injected IP with 2 × 10 7 of the remaining PBMCs.

Techniques: Laser Capture Microdissection, RNAscope, Staining, Clinical Proteomics