Journal: Nature Communications

Article Title: Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease

doi: 10.1038/s41467-020-16115-w

Figure Lengend Snippet: a Gating strategy in screening experiment using the panel of TCR Vβ antibodies. First, CD3 lymphocyte were separated into CD4 + and CD8 + populations using monoclonal antibodies (CD8 + : Pe-Cy7, CD4 + : PerCP). Next, the samples were stained with Vβ specific antibody cocktails included in IO Test Beta Mark TCR beta Repertoire Kit (Beckman-Coulter Immunotech, USA). The β variable chain family was determined based on FITC and PE positivity from CD4 + and CD8 + populations according to the manufacturer’s instruction. Vβ20 clone was detected from total CD4 + T cells (52.9%, middle panel) and total CD8 + T cells (1.74%, right). b Flow cytometry Vβ screening results from the index patient’s peripheral blood sample. T cell clonality with antibodies which target Vβ region of TCR was analysed of CD4 + T cells. The increased distribution suggests that the cells have large T cell clone. c Increased Vβ20 bearing clonotype over time in the index patient’s CD4 + T cells. Source data are provided as a Source data file. d T cell repertoire of FACS-sorted CD4 + Vβ20+ and CD8 + T cells analysed with TCRβ deep sequencing (Adaptive Biotechnologies). The TCRBV30-01 clone was detected in the CD4 + Vβ20+ fraction, but not in the CD8 + fraction. e Multicolor flow cytometry was applied to identify the immune phenotype of HSCT donor and index patient’s memory T cell subtypes. Central memory (CM), naïve, effector memory (EM), and terminal effector memory (TEMRA) cells. f The relative proportion of granzyme B positive (GrB + ) CD4 + T cells and GrB + CD8 + T cells in index patient. Index patient’s PBMCs were stained with anti-CD45, −CD3, −CD4, and −CD8 (surface markers), and then GrB stained after fixation and permeabilization. Stained cells were analyzed using FACSVerse.

Article Snippet: Thereafter, HIER, peroxide and protein block were repeated, followed by application of anti-CD8 (1:500, Clone: C8/144B, Cat#: BSB 5174, Lot#: 5174JDL05, BioSB) primary antibody, HRP-conjugated secondary antibody diluted 1:3 with washing buffer and TSA 555 (PerkinElmer).

Techniques: Staining, Flow Cytometry, Sequencing

Journal: Nature Communications

Article Title: Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease

doi: 10.1038/s41467-020-16115-w

Figure Lengend Snippet: Somatic mutations discovered in CD4 + T cells in the index patient, detected from 2013 sample.

Article Snippet: Thereafter, HIER, peroxide and protein block were repeated, followed by application of anti-CD8 (1:500, Clone: C8/144B, Cat#: BSB 5174, Lot#: 5174JDL05, BioSB) primary antibody, HRP-conjugated secondary antibody diluted 1:3 with washing buffer and TSA 555 (PerkinElmer).

Techniques: Mutagenesis

Journal: Nature Communications

Article Title: Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease

doi: 10.1038/s41467-020-16115-w

Figure Lengend Snippet: a Locations of mTOR, TLR2 , and NFκB2 somatic mutations. Linearized structure of MTOR, NFκB2 , and TLR2 presenting the location of somatic mutations. mTOR P2229R mutation is located in the kinase domain, NFκB2 P882Q in the C-terminus, and TLR2 W558L between LRR (Leucine-rich repeats) domain and transmembrane (TM) domain. b A heterozygous mTOR mutation (G to C, P2229R ) was detected in CD4 + T cells by Sanger sequencing. c Variant allele frequencies (VAFs) of mTOR P2229R , NFκB2 P882Q , and TLR2 W558L mutations in the index patient’s CD4 + T cells over time as measured with amplicon sequencing. Source data are provided as a Source data file. d VAFs (%) of mTOR P2229R mutation from the index patient’s skin, liver, and eyes biopsy. e Immunofluorescence staining indicated CD3 + CD4 + and CD3 + CD8 + T cell infiltration in the skin. Paraffin embedded skin biopsy from the index patient was sectioned and stained with antibody specific human CD3 (cyan), CD4 (green), and CD8 (red). White arrows indicate infiltrated CD3 + CD4 + or CD3 + CD8 + T cells. Original imaging magnification: 20×, the figure has been further zoomed to 25× for visualization. Scale bar: 50 μm.

Article Snippet: Thereafter, HIER, peroxide and protein block were repeated, followed by application of anti-CD8 (1:500, Clone: C8/144B, Cat#: BSB 5174, Lot#: 5174JDL05, BioSB) primary antibody, HRP-conjugated secondary antibody diluted 1:3 with washing buffer and TSA 555 (PerkinElmer).

Techniques: Mutagenesis, Sequencing, Variant Assay, Amplification, Immunofluorescence, Staining, Imaging

Journal: Nature Communications

Article Title: Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease

doi: 10.1038/s41467-020-16115-w

Figure Lengend Snippet: Somatic MTOR and NFκB2 mutations validated by amplicon sequencing in the index patient.

Article Snippet: Thereafter, HIER, peroxide and protein block were repeated, followed by application of anti-CD8 (1:500, Clone: C8/144B, Cat#: BSB 5174, Lot#: 5174JDL05, BioSB) primary antibody, HRP-conjugated secondary antibody diluted 1:3 with washing buffer and TSA 555 (PerkinElmer).

Techniques: Amplification, Sequencing

Journal: Nature Communications

Article Title: Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease

doi: 10.1038/s41467-020-16115-w

Figure Lengend Snippet: Real-time cell analysing (RTCA) systems, xCELLigence TM , was applied to monitor real-time killing effect of primary fibroblasts obtained from the index patient and healthy donors. a Index patient’s primary fibroblasts were cultured as monolayers for 24 h to reach full confluence. Once confluent, the effector cells; NK-92 cell (positive control, 8:1), primary CD4 + T cells (4, 8, 16:1) and primary CD8 + T cells (16:1) with different ratios (effector cells:fibroblasts) were added to each well followed by co-culture (arrow indicates the point of adding the effector cells, 0 h). The control (black line, media) shows the impedance of the fibroblasts without any added effectors. The cell impedance was measured every 30 min for 21 h. The measured impedance was expressed as Cell Index with the normalization performed at time of addition of effector cells. b Visualization of monolayers of the primary fibroblast before and 21 h after addition of effector cells (8:1 for all effector cells). c CD4 + T cells and CD8 + T cells from healthy donors were added to healthy donors’ own fibroblasts ( n = 3). The cell impedance was measured for 21 h. Data is representative of three independent individuals. d Index patient’s fibroblasts were seeded with HLA1 and HLA2 antibodies (10 and 20 μg/mL). After CD4 + T cells (CD4 + T cells:fibroblasts = 8:1) were added the cell impedance was measured for 21 h. Dots represent mean values and error bars indicate range ( n = 2 for all conditions, technical duplicates). Source data are provided as a Source data file.

Article Snippet: Thereafter, HIER, peroxide and protein block were repeated, followed by application of anti-CD8 (1:500, Clone: C8/144B, Cat#: BSB 5174, Lot#: 5174JDL05, BioSB) primary antibody, HRP-conjugated secondary antibody diluted 1:3 with washing buffer and TSA 555 (PerkinElmer).

Techniques: Cell Culture, Positive Control, Co-Culture Assay

Journal: Bioactive Materials

Article Title: ADGRG1-targeted hypoxia preconditioned extracellular vesicles ameliorate intervertebral disc degeneration by delivering taurine to disrupt the oxidative stress feedback loop-driven ferroptosis in nucleus pulposus cells

doi: 10.1016/j.bioactmat.2026.02.029

Figure Lengend Snippet: Preparation and characterization of engineered ADGRG1-targeting and hypoxia-treated EVs. (A)Induced fit docking analysis of ADGRG1-binding peptide (A1TP, 7 peptides) and extracellular domain of ADGRG1 protein (PDB database: 7SF8). (B) Analysis of the binding of the A1TP to purified ADGRG1 proteins using a microscale thermophoresis (MST) binding assay. (C) Induced fit docking analysis of A1TP-PEG and extracellular domain of ADGRG1 protein. (D) The binding free energy between A1TP or A1TP-PEG and ADGRG1 were calculated using molecular dynamics simulations. Lower values indicate more stable interactions, with values less than or equal to −20 considered as stable binding modes. (E) Schematic illustration of the conjugating reaction between DSPE-PEG-Alkyne and A1TP. Schematic illustration of the fabrication of A1TP-HX-EVs through external modification by A1TP anchoring. Specific steps for the synthesis of DSPE-PEG-A1TP (DPA) are shown in . (F) FT-IR analysis showed the characteristic peaks of the DSPE-PEG-A1TP. The new triazole ring itself showed a characteristic C=N stretching vibration, a peak at 1538 cm −1 revealed the successful conjugation of A1TP. (G) H Nuclear magnetic resonance (NMR) spectra of DSPE-PEG-A1TP in D2O. The hydrogen signatures of the phenyl and phenol groups at 7.5-8.0 ppm confirmed the successful conjugation of DSPE to A1TP. (H) Western blot analysis verified the presence of three EV marker proteins (ALIX, TSG101, and CD81) and one EV negative marker (GM130) in EVs, HX-EVs, and A1TP-HX-EVs. (I) Transmission electron microscopy (TEM) images of EVs, HX-EVs and A1TP-HX-EVs. Scale bar, 200 nm. (J) Zeta potentials of EVs, HX-EVs and A1TP-HX-EVs, n = 3. Two-tailed unpaired Student's t-test was used for statistical analysis. ns, not significant. A two-tailed unpaired Student's t-test was used for statistical analysis. (K) Representative images of the spherical morphology and dispersion states of EVs, HX-EVs and A1TP-HX-EVs. Scale bar, 500 nm. (L) Size distributions of EVs, HX-EVs and A1TP-HX-EVs.

Article Snippet: Finally, the presence of the characteristic EV markers Alix (92880, Cell Signaling Technology), CD81 (56039, Cell Signaling Technology) and TSG101 (sc-7964, Santa Cruz Biotechnology) was confirmed by Western blot analysis.

Techniques: Binding Assay, Purification, Microscale Thermophoresis, Modification, Conjugation Assay, Nuclear Magnetic Resonance, Western Blot, Marker, Transmission Assay, Electron Microscopy, Two Tailed Test, Dispersion

Journal: Communications Biology

Article Title: Immune checkpoint blockade in triple negative breast cancer influenced by B cells through myeloid-derived suppressor cells

doi: 10.1038/s42003-021-02375-9

Figure Lengend Snippet: C57/Bl6 mice bearing E0771 tumors were treated with saline, FEC, oHSV-1, FEC + oHSV-1 or FEC + oHSV-1 + CP and tumors were harvested on day 7 ( n = 5 per group). Tumors were sectioned and stained with H&E for pathological analysis. Sections were then further stained with antibodies for CD3, CD4, CD8α, and FOXP3. a Representative images for tumors harvested on day 7. Each image shows a whole section of an individual tumor. b Whole tumor sections were scanned and quantified using HALO quantification software. Each symbol represents an individual mouse within that group. Two-tailed paired t test was used for statistical analyses. Error bars are representative of the standard deviation.

Article Snippet: Primary antibody staining was performed in five rounds: CD3 (1:600, Spring Bioscience Corp., M3074), PNAd (1:200, Biolegend, 120802), Pax5 (1:1000, Abcam, EPR3730), CD8 (1:200, Cell Signaling Technologies, 98941 S), CD11b (1:7500, Abcam, EPR1344).

Techniques: Saline, Staining, Software, Two Tailed Test, Standard Deviation