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