Journal: Cell

Article Title: Complement activation induces excessive T cell cytotoxicity in severe COVID-19

doi: 10.1016/j.cell.2021.12.040

Figure Lengend Snippet: Increased degranulation and cytotoxic potential of T cells from severe COVID-19 (A) Box and whisker (5–95 percentile) plots of SARS-CoV-2-specific IgG and IgA antibody levels detected in serum samples from mild (n = 15) and severe (n = 17) COVID-19 patients collected between day 10 and 14 post symptom onset. Wilcoxon test ∗∗ p < 0.01. (B) Linear regression analysis of TFH cell proportions (CyTOF cluster 7) determined in samples collected from mild (n = 8) and severe (n = 11) COVID-19 (cohort 1) during day 5 and 14 post-symptom onset and SARS-CoV-2-specific IgG and IgA serum levels. (C) Box and whisker (min − max) plots summarizing the intracellular granzyme B expression (unstimulated) of CD8 + T cells from PBMCs of mild (n = 21) and severe (n = 28) COVID-19 patients as well as controls (n = 21). Kruskal-Wallis & post hoc Dunn’s multiple comparison test ∗ p < 0.05, ∗∗∗ p < 0.001. (D) Box and whisker (5–95 percentile) plots summarizing the degranulation capacity of CD8 + T cells from PBMCs of mild (n = 21) and severe (n = 28) COVID-19 patients as well as controls (n = 20) defined by their increase of cell surface CD107a expression upon stimulation with anti-CD16 antibody-coated or isotype-coated beads. Multiple Mann-Whitney test ∗ p < 0.05, ∗∗∗ p < 0.001. (E) Scatter plot of the degranulation capacity from PBMCs of severe COVID-19 patients (n = 12) upon stimulation with spike-protein-coated beads pre-incubated with control serum, with spike-protein-coated beads pre-incubated with COVID-19 serum or with anti-CD16 antibody-coated beads. Kruskal-Wallis and post hoc Dunn’s multiple comparison test ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (F) Enrichment of CD137 + CD69 + cells in activated CD16 − (CD38 + CD16 − ) and activated CD16 + (CD38 + CD16 + ) over the total CD8 + T cell compartment upon restimulation of PBMC samples from mild (n = 5) and severe (n = 7) COVID-19 patients with a SARS-CoV-2 peptide pool. Enrichment was calculated by dividing the proportions of CD137 + CD69 + T cells in non-activated CD16 − (CD38 + CD16 − ) and activated CD16 + (CD38 + CD16 + ) by the proportions of total CD8 + T cells. Friedmann test & post hoc Dunn’s multiple comparison test. ∗ p < 0.05, ∗∗ p < 0.01. (G) Box and whisker (5–95 percentile) plots summarizing the normalized release of CXCL8 and CCL2 by primary lung endothelial cells co-cultured with CD8 + T cells enriched from PBMCs of mild (n = 6) and severe (n = 5) COVID-19 patients as well as non-infected controls (n = 5) upon stimulation with ConA or anti-CD16 antibody-coated beads. Wilcoxon test ∗ p < 0.05. (H) Endothelial cell resistance upon stimulation with ConA alone (n = 5) or additional co-culture with CD8 + T cells enriched from PBMCs of mild (n = 6) and severe (n = 5) COVID-19 patients as well as non-infected controls (n = 5). Kruskal-Wallis test ∗ p < 0.05. (I) Representative immunofluorescence staining of CD3 (green) and CD16 (red) in autopsy lung tissues of patients without lung pathology, with COVID-19, ARDS, or influenza pneumonia. (J) Quantification of CD3/CD16 double-positive T lymphocytes per mm 2 in the autopsy cohorts of deceased patients without lung pathology (n = 4) compared with COVID-19 (n = 13), ARDS (n = 8) and influenza pneumonia (n = 6). The COVID-19 cohort was separated into early stage (death after 7–14 days after first symptoms), mid stage (15–30 days after symptom onset), and late stage (>35 days). One-way ANOVA; ∗∗ , p < 0.01.

Article Snippet: CD69 162Dy (FN50) , Fluidigm , Cat# 3162001B; RRID: N/A.

Techniques: Whisker Assay, Expressing, Comparison, MANN-WHITNEY, Incubation, Control, Cell Culture, Infection, Co-Culture Assay, Immunofluorescence, Staining

Journal: Cell

Article Title: Complement activation induces excessive T cell cytotoxicity in severe COVID-19

doi: 10.1016/j.cell.2021.12.040

Figure Lengend Snippet: Representative plots of gating strategy, related to Figure 3 F (A) Gating of CD137 + CD69 + of CD8 + CD16 + CD38 + T cells, also discriminationg between CD8 low and CD8 high cells, as shown in Figure 3 F. Both, CD8 low & CD8 high expressing T cells from patients with severe COVID-19 contain high proportions of CD16 + CD38 + cells.

Article Snippet: CD69 162Dy (FN50) , Fluidigm , Cat# 3162001B; RRID: N/A.

Techniques: Expressing

Journal: Cell

Article Title: Complement activation induces excessive T cell cytotoxicity in severe COVID-19

doi: 10.1016/j.cell.2021.12.040

Figure Lengend Snippet:

Article Snippet: CD69 162Dy (FN50) , Fluidigm , Cat# 3162001B; RRID: N/A.

Techniques: Purification, Functional Assay, Blocking Assay, Recombinant, Lysis, Cell Isolation, Staining, Positive Control, Virus, Software, Derivative Assay

Journal: Cell reports

Article Title: Consumption of fish oil high-fat diet induces murine hair loss via epidermal fatty acid binding protein in skin macrophages

doi: 10.1016/j.celrep.2022.111804

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Hamster monoclonal anti-mouse CD69 - 143Nd , Fluidigm , Cat#3143004B; RRID:AB_2827881.

Techniques: Purification, Recombinant, Activation Assay, SYBR Green Assay, Reverse Transcription, Detection Assay, Enzyme-linked Immunosorbent Assay, Selection, Software

Journal: bioRxiv

Article Title: HIV-specific CD8 + T-cells in tonsils express exhaustive T RM -like signatures

doi: 10.1101/2021.11.04.467061

Figure Lengend Snippet: A . Blood and tonsil mononuclear cells pregated on CD8 T-cells by flow cytometry for perforin and granzyme B. B . Expression intensity of CD69, CD103, CXCR5, CD127, PD-1, Perforin and granzyme B in multidimensional tSNE space for CD8 + T-cells, gated from total CD45 + T-cells. Peripheral blood (blue), tonsil tissue (orange). C . Percentage of blood and tonsil CD8 + T-cells expressing indicated markers. D . Schematic of protocol for isolate of HIV + CD8 + T cells from peripheral blood and tonsil tissue by scRNA-seq using Seq-Well v3 (top). UMAP of CD8 + T cells coloured by tissue source (bottom). E . Heatmap of z-scored gene expression of CD8 + T cells illustrating differentially expressed genes between blood and tonsil. Selective specifically expressed genes are marked alongside. F . Violin plots showing selected genes for cytolytic, transcription factors, tissue-resident memory and immune suppressive markers, expressed in CD8 + T cells. FDR-adjusted p<0.05; full results can be found in Supplementary Table S1. G . Fluorescent immunohistochemistry of whole tonsil section (left) from HIV uninfected donor shown by CD8, CD4 and merged panels (middle) with quantification using 10 unrelated areas identified outside follicles (outside GCs) and within follicular germinal centers ( inside GCs). P-values by Kruskal-Wallis multi comparisons.

Article Snippet: Exactly 4μm sections were cut, deparaffinized and stained with the following unlabelled primary antibodies: CD8 (clone: C8/144B, Dako), CD4 (clone: 4B12, Dako), p24 (clone: Kal-1, Dako), CXCR5 (clone: MU5UBEE, Thermofisher Scientific), Granzyme B (clone: 23 H8L20, Thermofisher Scientific), CD103 (clone: NBP1-88142, Novus Biologicals) and CD69 (clone: 15B5G2, Novus Biologicals).

Techniques: Flow Cytometry, Expressing, Gene Expression, Immunohistochemistry

Journal: bioRxiv

Article Title: HIV-specific CD8 + T-cells in tonsils express exhaustive T RM -like signatures

doi: 10.1101/2021.11.04.467061

Figure Lengend Snippet: A . Percentage of blood (circles) and tonsil (triangles) CD8 + T cells expressing CD69, CD103, CXCR5, CD27, CD127, PD-1, granzyme B and perforin markers between HIV − and HIV + . Red data point indicates viremic (detectable HIV RNA) and red/black data point indicates suppressed (undetectable HIV RNA). P-values calculated using ordinary one-way ANOVA with horizontal bars representing median values with the level of significance indicated with p-value. B . Representative FACS plot of CD8 + T-cells from HIV-(grey triangles) and HIV + (red triangles includes both ART suppressed and viremic individuals) tonsils for co-expression of CD69/CD103 and PD-1/CD127 with cumulative data shown with horizontal bars representing median values and p-values by Kruskal-wallis multiple comparisons. C . CD8, CD69, CD103 fluorescent immunohistochemistry of whole tonsil sections zoomed in at individual GCs for three independent donors from HIV − (left), HIV + ART + (middle) and HIV + ART − (right) with individuals markers shown. D . Same as in C but for CD8, HIV-p24 and PD-1.

Article Snippet: Exactly 4μm sections were cut, deparaffinized and stained with the following unlabelled primary antibodies: CD8 (clone: C8/144B, Dako), CD4 (clone: 4B12, Dako), p24 (clone: Kal-1, Dako), CXCR5 (clone: MU5UBEE, Thermofisher Scientific), Granzyme B (clone: 23 H8L20, Thermofisher Scientific), CD103 (clone: NBP1-88142, Novus Biologicals) and CD69 (clone: 15B5G2, Novus Biologicals).

Techniques: Expressing, Immunohistochemistry

Journal: bioRxiv

Article Title: HIV-specific CD8 + T-cells in tonsils express exhaustive T RM -like signatures

doi: 10.1101/2021.11.04.467061

Figure Lengend Snippet: A . Representative FACS plot of the gating strategy of CD8 + T-cells from tonsil cells to detect naïve, T N (CCR7 + CD45RA + ), central memory (T CM , CCR7 + CD45RA − ), effector memory (T EM , CCR7 − CD45RA − ) and T EMRA (CCR7 − CD45RA + ). B . Different memory subset distribution within blood and tonsil CD8 + T-cells for central memory, T CM (blue), Effector memory, T EM (red), transitional, T EMRA (orange), T Naive (grey) with cumulative memory subset distribution of CD8 + T cells for blood (circles, left) and tonsil (triangles, right). C . Distribution of blood central memory (T CM ), transitional memory (T EMRA ), effector memory (T EM ) and naïve subsets within CD127, CD69, PD-1, perforin and granzyme B expressing CD8 + T-cells cumulative for all study participants in HIV − (grey) and HIV + (red). D . Same as in C but showing data from tonsil CD8 + T-cells. P-values calculated using ordinary one-way ANOVA with horizontal bars representing median values with the level of significance indicated above. E . The frequency of CD103, CD69, CD127, perforin, and granzyme B (Granz B) cells measured on PD-1 + (left) and PD-1 − (right) CD8 + T-cells from blood in HIV + (red) and HIV − (grey) individuals. F . Same as in E but showing data from tonsil CD8 + T-cells. P-values calculated using Paired Student’s t test. Horizontal bars represent median values.

Article Snippet: Exactly 4μm sections were cut, deparaffinized and stained with the following unlabelled primary antibodies: CD8 (clone: C8/144B, Dako), CD4 (clone: 4B12, Dako), p24 (clone: Kal-1, Dako), CXCR5 (clone: MU5UBEE, Thermofisher Scientific), Granzyme B (clone: 23 H8L20, Thermofisher Scientific), CD103 (clone: NBP1-88142, Novus Biologicals) and CD69 (clone: 15B5G2, Novus Biologicals).

Techniques: Expressing

Journal: bioRxiv

Article Title: HIV-specific CD8 + T-cells in tonsils express exhaustive T RM -like signatures

doi: 10.1101/2021.11.04.467061

Figure Lengend Snippet: A . Representative flow plots showing CMV-(top) and HIV-specific (bottom) tetramer stains of blood (left) and tonsil tissue (right) from the same participant. B . Heat map showing expression frequencies for the indicated markers among CD8 + T cells from CMV tetramer and HIV tetramer specific CD8 + T-cells in blood (top) and tonsil (bottom) gated CD8 + T-cells with frequencies for each tetramer population indicated in the bar below from blue (0%) to red (100%). C . Frequencies of CD69, CD103, PD-1 and CD127 from HIV-, CMV-, and non-specific (‘CD8’) CD8 + T-cells within blood (left) and tonsil (right) tissue. P-values calculated using ordinary one-way ANOVA with horizontal bars representing median values with the level of significance indicated above.

Article Snippet: Exactly 4μm sections were cut, deparaffinized and stained with the following unlabelled primary antibodies: CD8 (clone: C8/144B, Dako), CD4 (clone: 4B12, Dako), p24 (clone: Kal-1, Dako), CXCR5 (clone: MU5UBEE, Thermofisher Scientific), Granzyme B (clone: 23 H8L20, Thermofisher Scientific), CD103 (clone: NBP1-88142, Novus Biologicals) and CD69 (clone: 15B5G2, Novus Biologicals).

Techniques: Expressing

Journal: bioRxiv

Article Title: HIV-specific CD8 + T-cells in tonsils express exhaustive T RM -like signatures

doi: 10.1101/2021.11.04.467061

Figure Lengend Snippet: A . Workflow of single-cell RNA sequencing from HIV infected tonsil tissue isolated CD8 + T-cells pre-sorted on HIV-, CMV- and ‘non-specific’ CD8 + T-cells from HIV infected participants indicated in Table S2. B . Dimensionality reduction using tSNE on scRNA-Seq cells coloured by Louvain cluster (top), participant ID (middle), and tetramer specificity (bottom). C . Heatmap of z-scored gene expression of top differentially expressed genes (t-test) between Louvain clusters from scRNA-seq data with cells grouped by Louvain cluster, genes grouped by hierarchical clustering (full gene lists in supplemental Table S3). D . Gene set enrichment scores for each of the 4 Louvain clusters (0-3) shown for ‘ T RM ’ , ‘Exhaustion’ , ‘proliferation’ and ‘activation’ [ , ] published gene lists. E . Heatmaps of z-scored gene expression of top differentially expressed genes (t-test) between single cells with high and low normalized MFI values of CD69, CD103, PD-1 and CD127. Selected genes labelled in plot, full gene lists in Table S4-S5. F . Gene lists from (E) scored against the published gene lists as indicated in D.

Article Snippet: Exactly 4μm sections were cut, deparaffinized and stained with the following unlabelled primary antibodies: CD8 (clone: C8/144B, Dako), CD4 (clone: 4B12, Dako), p24 (clone: Kal-1, Dako), CXCR5 (clone: MU5UBEE, Thermofisher Scientific), Granzyme B (clone: 23 H8L20, Thermofisher Scientific), CD103 (clone: NBP1-88142, Novus Biologicals) and CD69 (clone: 15B5G2, Novus Biologicals).

Techniques: RNA Sequencing, Infection, Isolation, Gene Expression, Activation Assay

Journal: STAR Protocols

Article Title: Mass cytometry staining for human bone marrow clinical samples

doi: 10.1016/j.xpro.2022.101163

Figure Lengend Snippet: Surface antibody master mix

Article Snippet: Anti-Human CD69 (FN50)-144Nd—100 Tests (1:100) , Fluidigm , Cat#3144018B.

Techniques:

Journal: STAR Protocols

Article Title: Mass cytometry staining for human bone marrow clinical samples

doi: 10.1016/j.xpro.2022.101163

Figure Lengend Snippet:

Article Snippet: Anti-Human CD69 (FN50)-144Nd—100 Tests (1:100) , Fluidigm , Cat#3144018B.

Techniques: Recombinant, Saline, Red Blood Cell Lysis, Staining, Blocking Assay, Antibody Labeling, Software, Cell Counting, Cytometry

Journal: Science Advances

Article Title: Nutrient availability regulates the secretion and function of immune cell–derived extracellular vesicles through metabolic rewiring

doi: 10.1126/sciadv.adj1290

Figure Lengend Snippet: ( A and B ) M2-Mφs were induced by IL-4/TGF-β (20 ng/ml each), and qPCR analysis was conducted to measure the expression of the M2 gene ( Arg1 , Mrc1 , and TGF- β) expression in M2-Mφs treated with EV GLN− (20 μg/ml) or EV GLN+ (20 μg/ml) for 48 hours ( n = 3; ***P < 0.001, **P < 0.01, ## P < 0.01, ### P < 0.01 versus the M2 group). ( C and D ) qPCR analysis of chemokine gene expression ( Ccl2 and Cxcl2 ) in THP-1 monocytes treated with EV GLN− preparations or EV GLN+ preparations (20 μg/ml) for 24 hours ( n = 3; ***P < 0.001, **P < 0.01 versus the “CON” group). ( E and F ) Chemotaxis evaluation of (E) conditioned culture medium from EV pretreated THP-1 cells and (F) EVs from splenocytes using a Transwell system, and the migrated cells in the lower chamber were counted using FCA ( n = 3; **P < 0.01, *P < 0.05 versus the CON group). ( G and H ) Mouse splenocytes were treated with ConA or ConA plus EV GLN− or EV GLN+ (20 μg/ml) for 72 hours, and the populations of activated CD4 + T cells and (CD3 + CD4 + CD69 + ) activated CD8 + T cells (CD3 + CD8 + CD69 + ) were determined by FCA ( n = 3). ( I and J ) Evaluation of immune responses in mice ( n = 5) intravenously injected with EV GLN− or EV GLN+ (30 μg/mouse) for 4 hours and immune cell populations (F4/80 + Mφs, Ly6C + monocytes, and Ly6G + neutrophils) in the spleen were analyzed using FCA ( **P < 0.01, *P < 0.05 versus the CON group).

Article Snippet: After treatment, cells from each group were collected and stained with fluorescein isothiocyanate (FITC)–conjugated anti-mouse CD3e (553061, BD, Brea, CA, USA, USA), peridinin chlorophyll protein (PerCP)/Cyanine5.5-conjugated anti-mouse CD4 (100434, BioLegend), phycoerythrin (PE)–Cy7–conjugated anti-mouse CD8a (552887, BD), and allophycocyanin (APC)–conjugated anti-mouse CD69 (APC-65105, Proteintech, Wuhan, China) for 30 min. After washing with PBS, the stained cells were analyzed using a flow cytometer (LSRFortessa, BD Biosciences).

Techniques: Expressing, Gene Expression, Chemotaxis Assay, Injection