Journal: Journal of Immunology Research

Article Title: Common Variable Immunodeficiency and Circulating T FH

doi: 10.1155/2016/4951587

Figure Lengend Snippet: Expression of costimulatory molecules (CD40L and ICOS) in PHA+IL-2 stimulated PBMC from CVID patients and N.

Article Snippet: Anti-CD4 (clone RPA-T4), CD25 (clone M-A251), CD45RO (clone UCHL1), CXC-chemokine receptors, 5 (CXCR5) (CD185, clone RF8B2) and 7 (CCR7, Clone 3D12), CD19 (clone HIB19), CD27 (clone M-T271), inducible T cell costimulator (ICOS) (CD278, clone DX29), CD40 ligand (CD40L) (CD154, clone TRAP1), and programmed-death 1 (PD-1) (CD279, clone MIH4) antibodies were purchased from BD Pharmingen, BD, and eBioscience (San Diego, CA, USA).

Techniques: Expressing

Journal: bioRxiv

Article Title: Synthetic antigen-presenting cells reveal the diversity and functional specialisation of extracellular vesicles composing the fourth signal of T cell immunological synapses

doi: 10.1101/2021.05.29.445691

Figure Lengend Snippet: (A) Representative time-lapse confocal microscopy showing the interaction between TH and BSLB and the active transfer of CD40L + synaptic stamps to BSLB (white arrowheads), which are left behind after the interaction resolves. CD40L was tracked using 1 µg/mL of anti-CD40L clone 24-31 AF647. (B) FCM analyses of vesicular transfer to BSLB (teal histograms) from TH (grey histograms). BSLB were reconstituted with increasing densities of α-CD3ε Fab and 200 molec./µm 2 of ICAM1 and 20 molec./µm 2 of CD40. Vertical lines indicates the mode of cells and BSLB in the condition with no α-CD3 Fab (null). (C) After conjugate separation by cold, tSV are released from BSLBs with the use of 50 mM EDTA. (D) Elution results in the release of TCR + , CD40L +, and CD81 + , and to a lesser extent CD63 + , from the surface of BSLB as measured by FCM staining comparing null BSLB (black histograms), with BSLBs prior to (green), and after elution (violet). (E) Transmitted electron microscopy of eluates reveals the presence of tSV (white arrows) and small soluble proteins. Scale bar= 200 nm. (F) Representative NanoFCM analyses showing the detection of TCRαβ + and CD81 + events in isolated EVs (left panel), and the eluted material from Null BSLB (middle panel) and α-CD3 + BSLB (tSV, right panel). (G) overlaid histograms showing TCRαβ expression on CD81 + vesicles from EVs (yellow) and tSV (light blue) compared to double negatives (grey histogram). (H) Left: Immunoblot for comparison of CD40L, CD81, Alix and TSG101 in the EVs or eluted tSV deriving from 5 × 10 6 of either cells or eluted BSLB, respectively. BSLB were previously cocultured with TH cells at 1:1 ratio. Whole cell lysate represents a total of 2.25 × 10 5 cells. Right: CD40L levels in eluted fractions (tSV) and steadily released EVs as measured by immunoblot. Shown is CD40L corrected MFI (cMFI) normalised to TSG101 cMFI. Normality was determined using Shapiro-Wilk test and statistical significance was determined by two-tailed unpaired t-test (D) and by unpaired, two-tailed Mann-Whitney test (H). *p≤0.05, **p≤0.005, ***p≤0.0005, ****p≤0.0001 and ns = not significant. Data represents mean ± SEM of n=2 (A,F,G) and n=4 independent experiments (B-E, H).

Article Snippet: Then, cells were stained with 1 µg/mL of wheat germ agglutinin (WGA) conjugated to CF568 (Biotium; #29077-1) and 1 µg/mL anti-CD154 (CD40L) clone 24-31 AF647 for 15 min at RT and in the dark.

Techniques: Confocal Microscopy, Staining, Electron Microscopy, Isolation, Expressing, Western Blot, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Synthetic antigen-presenting cells reveal the diversity and functional specialisation of extracellular vesicles composing the fourth signal of T cell immunological synapses

doi: 10.1101/2021.05.29.445691

Figure Lengend Snippet: (A) CD40 densities on the surface of human B cells isolated from either peripheral blood (CD19 hi HLA-DR + and CD40 + ) or palatine tonsils (also CXCR5 hi ). (B) Based on data in A, BSLBs were reconstituted with increasing densities of human CD40 and analysed for their capacity to instigate the release of CD40L + tSV. Quiescent and α-CD3/CD28-activated TH are compared. BSLBs presenting either no α-CD3ε or 1,000 molec./µm 2 were used in these experiments. (C) As in B, TH cells were incubated for 4h with BSLB and the density of CD40L compared to 1 h activated TH controls. (D) TSG101 expression by flow cytometry (left) and immunoblotting in quiescent and activated TH. (E and F) Comparison of CD40L + tSV transfer among different T cell types ( green: TH; blue: Treg-enriched; violet: CTL), and among T cell clones (E) responding to the influenza H3 HA 338-355 peptide presented in HLA-DRB1*09:01. (G) TIRFM images showing CD40L clustering within the synapses of T cell clones stimulated on SLB containing 30 molec./µm 2 of antigen, 200 molec./µm 2 of ICAM1 and 20 molec./µm 2 of CD40. Scale bar = 5 µm (A-H) After cell: BSLB conjugate dissociation FCM measurement was performed on BSLB to calculate the NST% of tSV positive for (H) TCR, (I) CD2, (J) CD28, (K) CD63, (L) CD81, (M) CD40L and (N) BST2 was measured on control (green) or BSLB presenting CD80, CD86, ICOSL (blue), CD58 (red), PD-L1 and PD-L2 (magenta), HIV-1 gp120 (grey), and OX40L and 4-1BBL (black) (all at 100 molec./µm 2 ). (O) Representative half-overlaid histograms showing CD40L + tSV transfer to ligand- and α-CD3ε-Fab-coated BSLB. Control BSLB were tailored to present ICAM1, CD40 and titrations of α-CD3ε-Fab. (P) NST% of CD4 to control (green) and HIV-1 gp120-presenting BSLBs (grey; red arrows indicate the loss of CD4 on TH). Normality was determined using Shapiro-Wilk test, and statistical significance was determined by multiple t-test to compare levels of CD40L + tSV transferred to BSLB across different CD40 densities, times, T cell populations (B-C, E-F), and increasing antigen densities (either α-CD3ε Fab or HLA-DRB1*09:01:HA complexes). α-CD3ε-Fab EC 50 and marker maximum transfer (T max ) were calculated using three to four parameters F-test (E, F). In D, Wilcoxon matched pairs signed rank test. P = 0.0312 was used to compare TSG101 expression between quiescent and activated cells. In H-P, multiple t-test comparing control BSLB presenting 200 molec./µm 2 of ICAM1, 20 molec./µm 2 of CD40 and α-CD3ε Fab (0 to 2,000 molec./µm 2 ) with BSLBs also presenting the selected group of proteins each at 100 molec./µm 2 . *p≤0.05, **p≤0.005, ***p≤0.0005, ****p≤0.0001 and ns = not significant. Data represents means ± SEM of n=4 (A-E and H-P) and n=3 (F, G) independent experiments.

Article Snippet: Then, cells were stained with 1 µg/mL of wheat germ agglutinin (WGA) conjugated to CF568 (Biotium; #29077-1) and 1 µg/mL anti-CD154 (CD40L) clone 24-31 AF647 for 15 min at RT and in the dark.

Techniques: Isolation, Incubation, Expressing, Flow Cytometry, Western Blot, Comparison, Clone Assay, Marker

Journal: bioRxiv

Article Title: Synthetic antigen-presenting cells reveal the diversity and functional specialisation of extracellular vesicles composing the fourth signal of T cell immunological synapses

doi: 10.1101/2021.05.29.445691

Figure Lengend Snippet: (A) Gating strategy for selecting single BSLB and T cells to measure the transfer of vesicular effector at the IS. BSLBs presenting α-CD3ε Fab (0 to 1,000 molec./µm 2 ), 200 molec./µm 2 of ICAM1 and 20 molec./µm 2 of CD40 were used in all experiments. (B-L) Percent Normalized Synaptic Transfer (NST%= [cGMFI BSLB /(cGMFI BSLB + cGMFI Cells )]*100) to BSLB from TH (green), Treg (blue) and CTL (violet). Ligands included (B) the antigen receptor heterodimer TCRαβ; (C) CD2; (D) TCR co-receptors (CD4 or CD8); (E) CD28; (F) CD45; (G) CD63; (H) CD81; (I) BST2; (J) CD38, (K) CD39; (L) CD73, and (M) CD40L. (N) Perforin (PRF1) transfer as background corrected GMFI is shown (null BSLB-subtracted). Representative histograms for most makers are shown on the right for TH (green) and CTL (violet). Statistical significance was determined by multiple t-test comparing tSV transfer across different α-CD3ε Fab densities and among different T cell populations. α-CD3-Fab EC 50 and the marker transfer maximum (T max ) were determined using F-test and three to four parameters fitting (H-J). *p 0.05, **p≤ .005, ***p≤0.0005, ****p≤0.0001 and ns = not significant. Data represent means ± SEM of n=4-10 independent experiments.

Article Snippet: Then, cells were stained with 1 µg/mL of wheat germ agglutinin (WGA) conjugated to CF568 (Biotium; #29077-1) and 1 µg/mL anti-CD154 (CD40L) clone 24-31 AF647 for 15 min at RT and in the dark.

Techniques: Marker

Journal: bioRxiv

Article Title: Synthetic antigen-presenting cells reveal the diversity and functional specialisation of extracellular vesicles composing the fourth signal of T cell immunological synapses

doi: 10.1101/2021.05.29.445691

Figure Lengend Snippet: (A-E) Data showing the transfer of tSV as measured by flow cytometry of BSLB after the dissociation of cell:BSLB conjugates and expressed as percent of the maximum transfer (T max %) observed in CD4 -edited controls. Heatmaps show T max % for different tSV populations, including those positive for (A) CD40L, (B) TCRαβ, (C) CD63, (D) CD81, and (E) BST2 on BSLB incubated with different CRISPR/Cas9-edited cells (targets shown in italic). BSLB contained 200 molec./µm 2 of ICAM-1, 20 molec./µm 2 of CD40 and four increasing densities of anti-CD3ε Fab (0-2,000 molec./µm 2 ). Normality was determined using Shapiro-Wilk test and statistical significance was determined by multiple t-test corrected for multiple comparisons (Holm-Sidak) and comparing tSV transfer to BSLB between CRISPR/Cas9 edited cells and CD4 -edited controls (depicted as % of CD4-edited T max ). *p≤0.05, **p≤0.005, ***p≤0.0005, ****p≤0.0001 and ns = not significant. (F-G) TIRFM imaging and quantification of total CD40L FI in the synapses formed by CRISPR/Cas9 genome-edited cells. (F) Median CD40L integrated density (i.e. total FI) in immunological synapses of CRISPR/Cas9-edited cells as % of the maximum observed. Each circle represents a donor. Means of medians were compared using Mixed-effect analysis with geisser-greenhouse correction and Fisher’s LSD test. (G) Top: Representative TIRFM images showing mature immunological synapses and the levels of centrally clustered CD40L. Bottom: diagram depicting the imaging of CD40L and other interfacial signals in IS formed between T cells and stimulating SLB. Data representative of n=6 (A-E) and 5 (F-G) independent experiments.

Article Snippet: Then, cells were stained with 1 µg/mL of wheat germ agglutinin (WGA) conjugated to CF568 (Biotium; #29077-1) and 1 µg/mL anti-CD154 (CD40L) clone 24-31 AF647 for 15 min at RT and in the dark.

Techniques: Flow Cytometry, Incubation, CRISPR, Imaging