Journal: Nature Communications

Article Title: Structural analysis reveals TLR7 dynamics underlying antagonism

doi: 10.1038/s41467-020-19025-z

Figure Lengend Snippet: a Flow-chart of the conversion from TLR7 agonists to antagonists. The chemical structures of these compounds are shown. b The binding mode of Cpd-1 to TLR7 site 1 in its activated form is shown on the left. TLR7 is shown in ribbon-and-stick representations. Two TLR7 protomers in the dimeric structure are painted salmon and aquamarine, respectively. Dashed lines indicate hydrogen bonds. On the right is a schematic view of Cpd-1 binding to site 1. The space inside the dimerization interface and the key derivation part of Cpd-1 are indicated by a red ellipse. c Dose–response curves for the inhibition of human TLR7 activity by Cpd-5, Cpd-6, Cpd-7 and HCQ (positive control) measured by reporter gene assay with R848 (200 nM) as stimulating agent. Data are representative tests with three replicates. d Dose–response relationship between Cpd-7 and R848 measured by human TLR7 reporter gene assay. TLR7-expressing cells were incubated with Cpd-7 (0–100 μM), together with 0–1000 μM of R848. Data are representative tests with three replicates. e TLR7 inhibition assay in primary human PBMCs. PBMCs were pretreated by DMSO, Cpd-6 or Cpd-7 for 3 h before 20 h stimulation by DMSO (none) or R848. Secreted IFN-α levels quantified by ELISA were shown. Dot plots of the data with mean ( n = 3 per group) are shown.

Article Snippet: After 30 min, R848 (human TLR7/8 ligand, synthesized at Sumitomo Dainippon Pharma, Japan) or CpG2006 (human TLR9 ligand, Hokkaido System Science Co., Ltd., Japan) added to each well, wherein each final concentration were adjusted to 200 nM R848 (human TLR7), 30 μM R848 (human TLR8), or 500 nM CpG2006 (human TLR9).

Techniques: Binding Assay, Inhibition, Activity Assay, Positive Control, Reporter Gene Assay, Expressing, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Vaccines

Article Title: In Vitro Characterization of the Innate Immune Pathways Engaged by Live and Inactivated Tick-Borne Encephalitis Virus

doi: 10.3390/vaccines9060664

Figure Lengend Snippet: Assessment of the optimal concentration of RLR or MyD88 inhibitors in PBMCs. PBMCs from 3 healthy donors were pre-incubated with RLR inhibitors (ALX and BX) for 1 h ( A ) or with MyD88 inhibitor (Pepinh-MYD) and its peptide control (Control) for 6 h ( B ). Afterwards, the cells were treated for 24 h with Poly I:C (0.5 μg/mL) or R848 (10 μg/mL). Following stimulation, the cells were lysed and changes in gene expression were analyzed by RT-qPCR.

Article Snippet: TLR7/8 ligand R848 (10 μg/mL) and poly I:C-HMW/LyoVec (0.5 μg/mL) (both from Invitrogen) were used as controls to assess the stimulation of the cell platforms.

Techniques: Concentration Assay, Incubation, Expressing, Quantitative RT-PCR

Journal: Vaccines

Article Title: In Vitro Characterization of the Innate Immune Pathways Engaged by Live and Inactivated Tick-Borne Encephalitis Virus

doi: 10.3390/vaccines9060664

Figure Lengend Snippet: Gene expression levels in PBMCs stimulated with I-TBEV or live TBEV in the presence of inhibitors. PBMCs from 3 healthy donors were pre-incubated with RLR inhibitors (ALX, 50 µg/mL, and BX, 2 µM) for 1 h ( A – C ) or with MyD88 inhibitor (Pepinh-MYD, 10 µM) and its peptide control (Control) for 6 h ( D – F ). Afterwards, the cells were treated for 24 h with Poly I:C (0.5 μg/mL), R848 (10 μg/mL), I-TBEV (0.24 µg/mL) or live virus (MOI 10). Following stimulation, the cells were lysed and changes in gene expression were analyzed by RT-qPCR. Results are from 3 replicates. Levels of significance: ns: p > 0.05; *: p ≤ 0.05 and ***: p ≤ 0.001. Absence of labels indicates non-significant differences.

Article Snippet: TLR7/8 ligand R848 (10 μg/mL) and poly I:C-HMW/LyoVec (0.5 μg/mL) (both from Invitrogen) were used as controls to assess the stimulation of the cell platforms.

Techniques: Expressing, Incubation, Quantitative RT-PCR

Journal: Nature Communications

Article Title: γ-secretase directly sheds the survival receptor BCMA from plasma cells

doi: 10.1038/ncomms8333

Figure Lengend Snippet: ( a ) Human purified B cells were activated for 5 days as indicated; IgG and sBCMA in the supernatant were quantified using ELISA. Combined data of three independent experiments (mean±s.e.m., P =0.0073, paired t -test). ( b ) PBMCs were stimulated with R848+IL-2 for 7 days. IgG and sBCMA in the supernatant were quantified using ELISA. Combined data of three independent experiments (mean±s.e.m., P =0.0227, paired t -test). ( c – e ) Human purified B cells were stimulated with CD40L+IL-21. ( c ) surface BCMA was measured using flow cytometry on unstimulated B cells, CD19 + CD38 − cells and CD19 + CD38 + cells. ( d ) Sorted CD38 + and CD38 − cells were cultured for another 24 h and the amount of shed sBCMA was measured using ELISA, combined data of two independent experiments. ( e ) Correlation between sBCMA release and surface expression of BCMA for a single replicate. ( f , g ) sBCMA was immunoprecipitated from supernatant of plasmacytoma cells ( f , lanes 1, 2), serum ( f , lane 3) and from supernatant of human purified B cells cultured with CD40L plus IL-21 ( f , lane 4) with anti-BCMA monoclonal antibodies (mAbs) A7D12.2 ( f , lanes 1 and 4) or C12A3.2 ( f , lane 2) or goat-anti-BCMA ( f , lane 3). Western blot analysis for BCMA ( f ) and silver staining of sBCMA immunoprecipitated from plasmacytoma supernatant ( g ) was performed. ( h ) The band at 6 kDa (from g ) and sBCMA obtained using immunoprecipitation were analysed with mass spectrometry. The aa sequences of BCMA and peptides identified after tryptic (blue) or chymotryptic (red) digestion are shown. No peptide was detected with a C-terminal aa that was not a site for either tryptic or chymotryptic cleavage, indicating that the precise cleavage site of γ-secretase within the membrane needs to be identified.

Article Snippet: For stimulation of PBMC and native B cells, human IL-21 (EBioscience, San Diego, CA), TLR7+8 ligand R848 (Sigma-Aldrich, St Louis, MO) and human IL-2 (R&D Systems) were used.

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cell Culture, Expressing, Immunoprecipitation, Western Blot, Silver Staining, Mass Spectrometry

Journal: Nature Communications

Article Title: γ-secretase directly sheds the survival receptor BCMA from plasma cells

doi: 10.1038/ncomms8333

Figure Lengend Snippet: ( a , b ) Human B cells were differentiated into Ig-secreting cells via CD40L+IL-21. ( a ) Release of sBCMA on treatment with DAPT or TAPI-1 was measured using ELISA. sBCMA release was normalized to the amount of sBCMA shed under vehicle conditions, which was set as 100%. Combined data of three independent experiments (mean±s.e.m.). ( b ) These activated primary human B cells were subgrouped on the basis of expression of CD27 and CD38. A high surface expression of BCMA was seen on the CD27 ++ CD38 + subset. Surface expression of BCMA was enhanced using DAPT treatment (1 μM), while TAPI-I (50 μM) had little effect. ( c , d ) Human PBMCs were stimulated with R848+IL-2 for 7 days. ( c ) Release of sBCMA on treatment with DAPT or TAPI-1 was measured using ELISA. sBCMA release was normalized to the amount of sBCMA shed under vehicle conditions, which was set as 100%. Combined data of three independent experiments (mean±s.e.m.). ( d ) High surface expression of BCMA was seen on the CD19 + CD38 + subset; this was further enhanced by DAPT (1 μM), while TAPI-I (50 μM) had little effect.

Article Snippet: For stimulation of PBMC and native B cells, human IL-21 (EBioscience, San Diego, CA), TLR7+8 ligand R848 (Sigma-Aldrich, St Louis, MO) and human IL-2 (R&D Systems) were used.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing