Journal: Scientific Reports

Article Title: Toll-like receptor 2 induced cytotoxic T-lymphocyte-associated protein 4 regulates Aspergillus -induced regulatory T-cells with pro-inflammatory characteristics

doi: 10.1038/s41598-017-11738-4

Figure Lengend Snippet: TLR2 regulates Aspergillus -induced regulatory T-cells with a T H 17-like phenotype. ( A ) IL-17A production after 7 days, IL-10 production after 24 hours and 7 days, regulatory T-cell (CD25 + FoxP3 + ) induction after 7 days, and IL-17A and RORγt expression within regulatory T-cells after 7 days in human PBMCs (n = 6 donors) stimulated with heat-inactivated A. fumigatus conidia (1 × 10 7 /mL) after 1 hour pre-incubation with 10 µg/mL αTLR2, or CoIgG. ( B , C ) Different subsets within regulatory T-cell (CD25 + FoxP3 + ) induction, defined as RORγt - , RORγt + , or RORγt + IL-17A + regulatory T-cells (CD25 + FoxP3 + ), after 7 days in human PBMCs (n = 6 donors) stimulated with heat-inactivated A. fumigatus conidia (1 × 10 7 /mL) after 1 hour pre-incubation with 10 µg/mL αTLR2, or CoIgG. Data are represented as mean ± SEM. ( D ) IL-17A production after 7 days, IL-10 production after 24 hours and 7 days (n = 14 donors), regulatory T-cell (CD25 + FoxP3 + ) induction after 7 days, and IL-17A and RORγt expression within regulatory T-cells after 7 days in human PBMCs (n = 9 donors) stimulated with heat-inactivated A. fumigatus conidia (1 × 10 7 /mL) in the presence or absence of 10 µg/mL P3C or FSL1. ( E , F ) Different subsets within regulatory T-cell (CD25 + FoxP3 + ) induction, defined as RORγt - , RORγt + , or RORγt + IL-17A + regulatory T-cells (CD25 + FoxP3 + ), after 7 days in human PBMCs (n = 6 donors) stimulated with heat-inactivated A. fumigatus conidia (1 × 10 7 /mL) in the presence or absence of 10 µg/ml P3C. Data are represented as mean ± SEM. Abbreviations: Af = Aspergillus fumigatus ; CoIgG = Control immunoglobulin ( G ); αTLR2 = anti-TLR2 antibody; P3C = Pam3Cys-SKKKK; FSL-1 = Pam2Cys-SKKKK. *p-value ≤ 0.05, **p-value ≤0.01, ***p-value ≤ 0.001.

Article Snippet: P3C (10 μg/mL) (Pam3Cys-SKKKK; TLR2/TLR1 heterodimer ligand, EMC microcollections, Tübingen, Germany), FSL-1 (1 μg/mL) (Pam2Cys-SKKKK; TLR2/TLR6 heterodimer ligand, EMC microcollections, Tübingen, Germany), and anti-TLR2 blocking antibody/control IgG (10 μg/mL) (eBioscience, Halle-Zoersel, Belgium) were used for pre-incubation and co-stimulation with heat-inactivated A. fumigatus conidia.

Techniques: Expressing, Incubation, Control

Journal: Scientific Reports

Article Title: Toll-like receptor 2 induced cytotoxic T-lymphocyte-associated protein 4 regulates Aspergillus -induced regulatory T-cells with pro-inflammatory characteristics

doi: 10.1038/s41598-017-11738-4

Figure Lengend Snippet: CTLA4 regulates Aspergillus -induced regulatory T-cells with aT H 17-like phenotype. ( A ) Percentage of CTLA4 expressing CD4 + T-cells and Mean fluorescence intensity (MFI) of CTLA4 staining following stimulation of human PBMCs (n = 13 donors) with heat-inactivated A. fumigatus conidia (1 × 10 7 /mL), 10 µg/mL P3C or a combination of both. ( B ) IL-17A concentration in culture supernatants of human PBMCs (n = 6 donors) stimulated with heat-inactivated A. fumigatus conidia (1 × 10 7 /mL) after 1 hour pre-incubation with 24 µg/mL Abatacept, or CoIgG. ( C ) Regulatory T-cell (CD25 + FoxP3 + ) induction after 7 days in human PBMCs (n = 7 donors) stimulated with heat-inactivated A. fumigatus conidia (1 × 10 7 /mL) after 1 hour pre-incubation with 24 µg/mL Abatacept, or CoIgG. T H 17 cell (IL-17A + ) induction after 7 days in human PBMCs stimulated with heat- inactivated A. fumigatus conidia (1 × 10 7 /mL) after 1 hour pre-incubation with 24 µg/mL Abatacept, or CoIgG. IL-17A + regulatory T-cell induction after 7 days in human PBMCs stimulated with heat- inactivated A. fumigatus conidia (1 × 10 7 /mL) after 1 hour pre-incubation with 24 µg/mL Abatacept, or CoIgG. Data are represented as mean ± SEM. Abbreviations: P3C = Pam3Cys-SKKKK; Af = A spergillus fumigatus ; CoIgG = Control immunoglobulin G. *p-value ≤ 0.05, **p-value ≤ 0.01,***p-value ≤ 0.001.

Article Snippet: P3C (10 μg/mL) (Pam3Cys-SKKKK; TLR2/TLR1 heterodimer ligand, EMC microcollections, Tübingen, Germany), FSL-1 (1 μg/mL) (Pam2Cys-SKKKK; TLR2/TLR6 heterodimer ligand, EMC microcollections, Tübingen, Germany), and anti-TLR2 blocking antibody/control IgG (10 μg/mL) (eBioscience, Halle-Zoersel, Belgium) were used for pre-incubation and co-stimulation with heat-inactivated A. fumigatus conidia.

Techniques: Expressing, Fluorescence, Staining, Concentration Assay, Incubation, Control

Journal: Frontiers in Immunology

Article Title: Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

doi: 10.3389/fimmu.2021.764390

Figure Lengend Snippet: CXCL8 production by bovine MØ in response to FSL-1, M. bovis BCG or MTB Ligand Stimulation. Bovine MØ of BS (heterozygous for TLR2 H326Q SNP) and HF MØ (homozygous for the wild-type TLR2 sequence) were stimulated with either FSL-1 at 100 ng mL -1 (A) , 19 kDa lipoprotein antigen (represents Rv3763 or Mb3789) (EMC microcollections, Germany) or infected with M. bovis BCG at MOI = 5 (B) for 24 hr. CXCL8 concentration in cell culture supernatant was assessed by ELISA. A total of n=8 animals per breed were stimulated with FSL-1 and n=6 animals per breed were infected with M. bovis BCG. Error bars represent standard deviation from the mean. Data was analysed with two-way ANOVA and Tukey’s post hoc comparison in GraphPad Prism V8 (GraphPad Inc., USA) (*=p<0.05, ***=p<0.001).

Article Snippet: In brief, SEAP HEK cells expressing TLR2 constructs, or PBMC-derived bovine MØ were specifically stimulated using 100 ng mL -1 diacylated FSL-1 (a TLR2/TLR6 antagonist; In vivo gen, USA); 1 mg mL -1 of Pam 3 CSK 4 (a TLR2/TLR1 antagonist, In vivo gen); 19 kDa lipoprotein antigen (represents Rv3763 or Mb3789) (EMC microcollections, Germany), M. bovis BCG (MOI of 10) or MTB H37Rv at (MOI of 5).

Techniques: Sequencing, Infection, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation, Comparison