Journal: Biomaterials

Article Title: Celastrol nanoemulsion induces immunogenicity and downregulates PD-L1 to boost abscopal effect in melanoma therapy.

doi: 10.1016/j.biomaterials.2020.120604

Figure Lengend Snippet: Scheme 1. Schematic illustration of the intratumorally injected celastrol nanoemulsion (CEL NE) simultaneously inducing immunogenic cell death (ICD) and PD-L1 downregulation, boosting the systemic abscopal effect on B16F10 bilateral tumor model. CEL NE i.t. injected in the subcutaneous tumor on one side continuously released CEL and induced tumor cells to expose calreticulin (CRT) and release HMGB1 as the tumor-associated antigens, which were engulfed by antigen-presenting cells (DC cells) and primed CD8+ T cells infiltration and activation. Meanwhile, CEL NE also effectively downregulated PD-L1 expression in tumor cells. The synergy of strong ICD and PD-L1 reduction activated the tumor immunosuppressive microenvironment and effector CD8+ T cells, giving potent tumor inhibition of both primary tumor and distant contralateral tumor as well as long-lasting systemic tumor suppression.

Article Snippet: Supernatant released HMGB1 was quantitated according to HMGB1 ELISA kit (orb409067 and orb406327, Biorbyt, Ltd.).

Techniques: Injection, Activation Assay, Expressing, Inhibition

Journal: Biomaterials

Article Title: Celastrol nanoemulsion induces immunogenicity and downregulates PD-L1 to boost abscopal effect in melanoma therapy.

doi: 10.1016/j.biomaterials.2020.120604

Figure Lengend Snippet: Fig. 1. Celastrol (CEL) induces ICD and down-regulates PD-L1 expression in melanoma in vitro and in vivo. (A) Chemical structure of CEL. (B) CEL triggered autophagy in melanoma cells. Mouse B16F10 or BPD6 melanoma cells were incubated with CEL (0.1–8 μM) for 12 h and then lysed for Western blot analysis of LC3B, whose subunit transition from LC3B I to LC3B II is a marker for activated autophagy. (C) Immunofluorescent imaging of CRT, HMGB1, and PD-L1 in mouse (B16F10 and BPD6) and human (M10 and A375) melanoma cells after treated with CEL at 1 μM; The treatment time was 4 h for CRT and 24 h for HMGB1 and PD-L1 detection. Cell nuclei were stained with DAPI. Scale bar indicates 20 μm. Each value was quantified in 5 randomly selected fields. Each sample was repeated 3 times. (D) Flow cytometry analysis of CRT+ melanoma cells after treated with CEL or positive control, doxorubicin (DOX) and mitoxantrone (MIT), for 4 h at their IC50. (E) The released HMGB1 in the cell culture medium 24 h after incubation with CEL, DOX or MIT at their IC50 doses (n = 4). (F) RT-PCR analysis of the PD-L1 mRNA levels in B16F10 and BPD6 cells after incubation with CEL, DOX or MIT at IC50 for 24 h (n = 6). (G) Western blot analysis of PD-L1 expression in tumors at 48 h after i.t. injection with CEL (0.15 mg/kg), DOX (0.01 mg/kg) or MIT (0.5 mg/kg) or i.p. injection with αPD-L1 (5 mg/kg). All data are shown as mean ± SD. *p < 0.05, **p < 0. 01, ***p < 0. 001, NS: not significant.

Article Snippet: Supernatant released HMGB1 was quantitated according to HMGB1 ELISA kit (orb409067 and orb406327, Biorbyt, Ltd.).

Techniques: Expressing, In Vitro, In Vivo, Incubation, Western Blot, Marker, Imaging, Staining, Flow Cytometry, Positive Control, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Injection

Journal: Biomaterials

Article Title: Celastrol nanoemulsion induces immunogenicity and downregulates PD-L1 to boost abscopal effect in melanoma therapy.

doi: 10.1016/j.biomaterials.2020.120604

Figure Lengend Snippet: Fig. 2. Characterization of celastrol nanoemulsion (CEL NE) and its ICD-induction and PD-L1 downregulation on melanoma. (A) The size distribution of CEL NE characterized by dynamic laser light scattering and transmission electron microscopy (insets). (B) The dose-dependent curve of CRT+-population of B16F10 cells incubated with free CEL or CEL NE for 4 h. The percentage of CRT+ cells was quantified as an average of 5 randomly selected fields. Each sample was repeated 3 times; see Supplementary Fig. S4 for the curves of other cells. (C–F) Analysis of in vivo ICD-induction and PD-L1 downregulation. The mice bearing B16F10 tumors received a single i.t. injection of CEL NE or free CEL (0.15 mg/kg) (n = 4) and were sacrificed after 48 h for analysis: (C) Representative immunofluorescent images of CRT, HMGB1 and activated DC markers (CD86, CD11c, and MHC II) in B16F10 tumors after i.t. injection of CEL NE or free CEL. Each value was quantified as an average of 5 randomly selected fields. Scale bar indicates 300 μm. (D) Statistic analysis of co-stimulatory markers, CD80 and CD86, in the draining lymph nodes by flow cytometry (n = 4). (E) Flow cytometry quantitation of PD-L1+ melanoma cell percentages (PD-L1+MART+) in the tumors. (F) RT-PCR analysis of PD-L1 mRNA levels in the tumors. (G) Western blot analysis of dose-dependent downregulation of NF-κB and PD-L1 in the tumors. The mice with tumors as above were i.t. injected with the indicated doses of CEL NE and analyzed after 48 h (n = 4). All data are shown as mean ± SD. *p < 0.05, **p < 0. 01, ***p < 0. 001, NS: not significant.

Article Snippet: Supernatant released HMGB1 was quantitated according to HMGB1 ELISA kit (orb409067 and orb406327, Biorbyt, Ltd.).

Techniques: Transmission Assay, Electron Microscopy, Incubation, In Vivo, Injection, Flow Cytometry, Quantitation Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Scientific Reports

Article Title: Somatic alterations compromised molecular diagnosis of DOCK8 hyper-IgE syndrome caused by a novel intronic splice site mutation

doi: 10.1038/s41598-018-34953-z

Figure Lengend Snippet: STAT3 phosphorylation analysis after stimulation. ( a ) Western blot analysis of whole cell lysates of PBMCs, unstimulated or 20 min. stimulated with 200 ng/ml IL6 or IL10. Expression of STAT3 phosphorylated at Y705 (pSTAT3) and total STAT3 (STAT3) of the two affected siblings and a healthy control was assessed; Actin as loading control. ( b ) Representative flow cytometric analysis showing diminished Y705-STAT3 phosphorylation after 20 min. stimulation with 200 ng/ml IL6 (solid line) versus unremarkable results after stimulation with 20 ng/ml IL10 (dotted line) and 10 ng/ml IL21 (dashed line) in lymphocytes of patient II.2 compared to unremarkable results in patient II.3 and a healthy control; filled gray area: unstimulated lymphocytes. ( c ) Flow cytometric analysis showing Y705-STAT3 phosphorylation after 20 min. stimulation with 20 ng/ml IL6 (solid line) or IL10 (dotted line) and 10 ng/ml IL21 (dashed line) comparable to healthy control in lymphocytes of one (representative of four) DOCK8-HIES patient. ( d ) Restored STAT3 phosphorylation after IL6 stimulation (solid line) in patient II.2 15 months after HSCT compared to unstimulated (filled gray area) and IL10-stimulated (dotted line) lymphocytes.

Article Snippet: Figure 4 DOCK8 expression analysis. ( a ) Western blot analysis of whole PBMC lysates shows DOCK8 expression in patient II.2 and not in patient II.3 with two different DOCK8 antibodies (immunogen indicated in brackets; aa: amino acid); Actin as a loading control.

Techniques: Phospho-proteomics, Western Blot, Expressing, Control

Journal: Scientific Reports

Article Title: Somatic alterations compromised molecular diagnosis of DOCK8 hyper-IgE syndrome caused by a novel intronic splice site mutation

doi: 10.1038/s41598-018-34953-z

Figure Lengend Snippet: Genetic analysis of DOCK8 . ( a ) T cell blast cDNA chromatograms show wildtype sequence in a healthy control, double peaks in patient II.2 and altered sequence in patient II.3. Both patients’ gDNA is homozygous for alteration c.4626 + 76 A > G; vertical black lines: 3′ junction of exon 36; black letters: wildtype; red letters: altered sequence. ( b ) Schematic model of affected region in DOCK8 gDNA and transcripts showing exon extension (dotted line) due to the novel splice site (*) introduced at c.4626 + 76 A > G; filled boxes: exons; horizontal line: intronic region. ( c ) Quantification of wildtype and altered transcripts in T cell blasts by ddPCR indicating percentages of wildtype (wt) of total DOCK8 transcripts in patient II.2, patient II.3 and healthy controls (HC). ( d ) ddPCR analysis of healthy controls (homozygous wt) and healthy carriers of a c.3120 + 1 G > T DOCK8 alteration resulting in exon 25 skipping (heterozygous). ( e ) Sashimi plot of RNA sequencing data based on GTEx samples , showing exon 32 skipping as a rare event; read counts accumulated over all samples. ( f ) Schematic model of wildtype and mutated minigene vectors. Sequence tags (PT1/PT2) flanked the minigene sequence to differentiate minigene transcripts from endogenous DOCK8 transcripts; filled boxes: exons; dotted line: exon extension; horizontal line: intronic regions; *: novel splice site. ( g ) The altered or physiologic transcription products of the minigene vectors were differentiated by size. Agarose gel with canonical splice site usage (378 nucleotide transcript) in cDNA of control PBMCs transfected with wildtype (Mini wt) and usage of the novel splice site (453 nucleotide transcript) in cDNA of PBMCs transfected with the mutated minigene vector (Mini mut); GFP- and mock-transfected as negative controls.

Article Snippet: Figure 4 DOCK8 expression analysis. ( a ) Western blot analysis of whole PBMC lysates shows DOCK8 expression in patient II.2 and not in patient II.3 with two different DOCK8 antibodies (immunogen indicated in brackets; aa: amino acid); Actin as a loading control.

Techniques: Sequencing, Control, RNA Sequencing, Agarose Gel Electrophoresis, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: Somatic alterations compromised molecular diagnosis of DOCK8 hyper-IgE syndrome caused by a novel intronic splice site mutation

doi: 10.1038/s41598-018-34953-z

Figure Lengend Snippet: DOCK8 expression analysis. ( a ) Western blot analysis of whole PBMC lysates shows DOCK8 expression in patient II.2 and not in patient II.3 with two different DOCK8 antibodies (immunogen indicated in brackets; aa: amino acid); Actin as a loading control. Full-length western blots are provided in the Supplementary Appendix (Supplementary Fig. ). ( b ) Flow cytometry of patient II.2 showed DOCK8 expression in majority of NK cells and T cells but no DOCK8 expression in B cells. All cell subsets of patient II.3 lack DOCK8 expression. Gray area: unstained; dashed line: isotype control; solid line: DOCK8 staining. ( c ) T cell subsets defined by naïve T cells (CCR7 + CD45RA + ), central memory T cells (CCR7 + CD45RA − ), effector memory T cells (CCR7 − CD45RA − ) and T EMRA cells (CCR7 − CD45RA + ) showed no DOCK8 expression in T EMRA and naïve T cells and DOCK8 expression in majority of central and effector memory T cells of patient II.2 compared to DOCK8 expression in all T cell subsets of a healthy control. ( d ) Sequencing of cDNA reveals double peaks in chromatograms of T and NK cells of patient II.2 indicating wildtype (black letters) and altered (red letters) transcripts. cDNA chromatogram of B cells shows only single peaks indicating altered transcripts.

Article Snippet: Figure 4 DOCK8 expression analysis. ( a ) Western blot analysis of whole PBMC lysates shows DOCK8 expression in patient II.2 and not in patient II.3 with two different DOCK8 antibodies (immunogen indicated in brackets; aa: amino acid); Actin as a loading control.

Techniques: Expressing, Western Blot, Control, Flow Cytometry, Staining, Sequencing

Journal: Scientific Reports

Article Title: Somatic alterations compromised molecular diagnosis of DOCK8 hyper-IgE syndrome caused by a novel intronic splice site mutation

doi: 10.1038/s41598-018-34953-z

Figure Lengend Snippet: Analysis of somatic alterations in DOCK8 . ( a ) Gating strategy to sort lymphocyte subsets according to their DOCK8 expression. PBMCs of patient II.2 were gated for lymphocytes and then DOCK8-negative cells into B cells (DOCK8 − CD19 + ) and non-B cells (DOCK8 − CD19 − ), and DOCK8-positive cells into T cells (DOCK8 + CD19 − CD3 + ) and NK cells (DOCK8 + CD19 − CD56 + ). ( b ) gDNA sequence of sorted cells of patient II.2 had a homozygous peak for the c.4626 + 76 A > G alteration (red letter) in T and B cells and a double peak with altered (red letter) and wildtype (black letter) sequence in NK cells. ( c ) gDNA sequence of unfixed and unpermeabilized PBMCs of patient II.2 sorted according to the lymphocyte subsets CD4 + and CD8 + T cells showing double peaks with altered (red letter) and wildtype (black letter) sequence in CD4 + T cells at positions c.4626 + 76 and c.4626 + 77 and at position c.4626 + 80 in CD8 + T cells.

Article Snippet: Figure 4 DOCK8 expression analysis. ( a ) Western blot analysis of whole PBMC lysates shows DOCK8 expression in patient II.2 and not in patient II.3 with two different DOCK8 antibodies (immunogen indicated in brackets; aa: amino acid); Actin as a loading control.

Techniques: Expressing, Sequencing