Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a ACE2+ EVs detected in human plasma samples of sero-negative controls (light blue), acute phase (dark green), and convalescent COVID-19 patients (green). One-tail t test (* p = 0.038, ** p = 0.0061 and ** p = 0.0016). Data are presented as mean values ± SEM. b Representative microflow vesiclometry (MFV) plots with gated ACE2+ EVs from sero-negative, acute phase and convalescent COVID-19 patients. c MFV detection of circulating ACE2 + EVs with CD63 + EVs in human plasma of convalescent COVID-19 patient samples (CSB-029 and CSB-023) (green line). Blue line is isotype IgG-negative control. d Flow profiles of ACE2 expression in HEK and HeLa parental control cells (Con, light blue line, ACE2 − ) and with ACE2 overexpression (ACE2, green line). e NanoSight NTA analysis of the sizes of HEK-derived ACE2 − (ev1Con) and ACE2 + (ev1ACE2) and HeLa-derived ACE2 − (ev2Con) and ACE2 + (ev2ACE2). f Immunoblots of HEK and HeLa (ACE2 − and ACE2 + ) EVs and cell lysates for ACE2, TSG101, CD63, CD81, GRP94 and loading control of the membrane proteins upon Ponceau staining. RIPA buffer and Bradford protein assay were used for cells/EVs lysis and protein measurement, respectively ( N = 1 experiment). g Cryo-EM images of HEK-derived EVs, ACE2 − (evCon, left) and ACE2 + (evACE2, right), stained with ACE2 (top) and CD81 (bottom). Scale bars = 100 nm. h Quantified counts of Apogee MFV-based total extracellular vesicles (EVs) and ACE2 + EVs ( N = 2 experiments with n = 6 technical replicates for total EV particles and n = 3 technical replicates for ACE2 + counts). Control EVs are in light blue and ACE2 + EVs in green. Data are presented as mean values +/− SD. i Overlay flow profiles of ACE2 positivity within CD63 + (left column) and CD81+ (right column) EVs isolated from HEK-ACE2 (top row) and HeLa-ACE2 (bottom row) cells, respectively ( n = 3 technical replicates). Light blue line for Control EVs and green line for ACE2 + EVs.

Article Snippet: Cells were blocked with mouse serum IgG (Sigma, 15381) for 10 min at room temperature and then incubated with specific antibodies; AF-647 mouse anti-human ACE2 (Clone # 535919) (R&D systems, FAB9332R), AF-488 mouse anti-human ACE2 (Clone # 171607) (R&D systems, FAB9333G) (0.4 μg/10 6 cells), AF-647 isotype control mouse IgG2b (Clone # 20102) (R&D systems, IC003R) or AF-488 isotype control mouse IgG2bAF488 (Clone # 20102) (R&D systems, IC003G) for 45 min on ice, followed by washing twice with 2% EV-free FBS/PBS.

Techniques: Clinical Proteomics, Negative Control, Expressing, Control, Over Expression, Derivative Assay, Western Blot, Membrane, Staining, Bradford Protein Assay, Lysis, Cryo-EM Sample Prep, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Tumor suppression of novel anti–PD-1 antibodies mediated through CD28 costimulatory pathway

doi: 10.1084/jem.20182359

Figure Lengend Snippet: Anti–PD-1 Abs significantly enhance proliferation of Ag-specific exhausted CD8 T cells. (A) Recovery of proliferation in HIV-specific CD8 T cells following stimulation with an HIV-derived peptide. Results from a representative experiment are shown and expressed as the percentage of CFSE-low CD8 T cells. 8–10 replicates were performed for each experimental condition. (B) Cumulative results from multiple CFSE experiments ( n = 2–6) are shown for the 10 anti–PD-1 Abs identified with antagonistic properties similar to pembrolizumab. Results have been generated assessing the proliferation of CD8 T cells specific to three HIV-derived peptides (FLGKIWPSYK restricted by A*0201 and RLRPGGKKK or RMRGAHTNDVK restricted by A*0301) in patients B08 and B09. For comparative purposes across multiple assays and the different anti–PD-1 Abs, the level of proliferation in the pembrolizumab-treated samples was set as a 100% reference. Pembrolizumab was used as a positive control, while untreated (Neg) or mouse IgG1 isotype control Ab was used as a negative control in each experiment. Graphs show the mean ± SD. ****, P < 0.0001 for all anti–PD-1 Abs relative to the IgG1 control (unpaired t test with Welch’s correction).

Article Snippet: The PD-1–PDL-1 biochemical protein–protein interaction assay was performed by preincubating PD-1 Fc coated Bio-Plex beads in the presence of 20 nM of anti–PD-1 Ab (clones 137F2, 135C12, and 136B4) or a mouse IgG1 isotype control for 1 h. Duplicate samples of biotinylated PD-L1 Fc protein (R&D Systems) were added in a concentration range from 0.08 to 10 nM to the preformed Ab–PD-1 complex and incubated for a further 4 h before removing unbound PDL-1 and staining beads with PE-labeled streptavidin.

Techniques: Derivative Assay, Generated, Positive Control, Control, Negative Control