Journal: bioRxiv

Article Title: DOCK8 regulates a mechanosensitive actin redistribution that maintains immune cell cohesion and protects the nucleus during migration

doi: 10.1101/2024.07.26.605273

Figure Lengend Snippet: Two representative cell outlines of activated WT and Dock8 KO T cells spontaneously migrating through a collagen matrix made of 2 mg/mL bovine type I collagen (no chemokine added), colour-coded by time with the direction of migration indicated (arrow). (b) Representative confocal microscopy images (max projection of z-stacks) of T cells migrating through collagen matrix as in (a). Examples of WT and Dock8 KO T cells (entangled or not) are shown with cell front and rear indicated. Fixed cells were stained for F-actin (phalloidin), tubulin (α-tubulin antibody), and nucleus (Hoechst). (c) Principal component analysis (PCA) of 20 extracted cell shape parameters from n = 211 WT and n = 293 KO T cells migrating in low and high collagen densities (1.5 and 4 mg/mL) after 4 hours. Data points are individual cells; example cell outlines of T cells classified as normal or entangled by PCA are shown. (d) Summary violin plots of the aspect ratio of cells analysed in (c) for each category. Data points: individual cells; solid lines: median; dotted lines: quartiles. (e) Summary plot of percent WT and Dock8 KO T cells entangled in collagen matrix (2 mg/mL) after 4 hours of migration. Data points: replicate T cell cultures from n = 4 mice; means are indicated. (f) Summary plot of percent live WT and Dock8 KO T cells in collagen matrix (1.5, 2, or 4 mg/mL) after 2 or 24 hours of migration. Data points: replicate T cell cultures from n = 4 mice; medians are indicated with quartiles (box) and min to max ranges (error bars). Statistical tests: Kruskal-Wallis ANOVA with Dunn’s multiple comparison test (d, e). * P < 0.05; *** P < 0.001; ns, non-significant.

Article Snippet: Two-step RT-qPCR: qPCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems) using 2 μL of cDNA per well combined with TaqMan Fast Advanced Master Mix, the TaqMan probe Mm01277042_m1 ( Tbp ) in VIC (endogenous control), and one of the following TaqMan probes, in FAM: Mm00613802_m1 ( Dock8 ), Mm01289583_m1 ( Wwtr1 ) (all reagents from Applied Biosystems).

Techniques: Migration, Confocal Microscopy, Staining, Comparison

Journal: bioRxiv

Article Title: DOCK8 regulates a mechanosensitive actin redistribution that maintains immune cell cohesion and protects the nucleus during migration

doi: 10.1101/2024.07.26.605273

Figure Lengend Snippet: Activated WT and Dock8 KO T cells expressing LifeAct-GFP and NLS- nTnG migrating in straight microchannels (width of 6 µm and height of 5 µm) coated with fibronectin, no chemokine added. Summary of individual cell mean speed (b), and representative examples acquired by epifluorescence microscope shown over time (c). Data is from 2 independent experiments, n = 361 (WT) and n = 179 (KO) cells; line: median. (d, e) Activated WT and Dock8 KO T cells expressing LifeAct-GFP and NLS-nTnG migrating through pillar forest microchannels (channel width of 6 µm, height of 5 µm, pillars of 8 µm x 8 µm) coated with fibronectin, no chemokine added. Summary of individual cell mean velocities (d), and representative examples acquired by epifluorescence microscope (e). Data is n = 572 (WT) and n = 1385 (KO) cells; line: median. (f, g, h) Activated WT and Dock8 KO T cells expressing LifeAct-GFP and NLS-nTnG migrating through microchannels with constrictions (channel width of 6 µm, height of 5 µm, constriction sizes 1.5–4 µm) coated with fibronectin, no chemokine added. Representative examples of cell retreating and cell passing acquired by epifluorescence microscope (f). Percent of cells passing through or retreating at constrictions (g), and summary of time spent passing through constrictions of different sizes, data points are individual cells, n = 18–57 cells per constriction size; line: median; dotted line at average passing time through 4 µm constrictions (h). Statistical tests: Mann-Whitney test (b, d); 2-way ANOVA (h). ** P < 0.01; *** P < 0.001; ns, non-significant.

Article Snippet: Two-step RT-qPCR: qPCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems) using 2 μL of cDNA per well combined with TaqMan Fast Advanced Master Mix, the TaqMan probe Mm01277042_m1 ( Tbp ) in VIC (endogenous control), and one of the following TaqMan probes, in FAM: Mm00613802_m1 ( Dock8 ), Mm01289583_m1 ( Wwtr1 ) (all reagents from Applied Biosystems).

Techniques: Expressing, Microscopy, MANN-WHITNEY

Journal: bioRxiv

Article Title: DOCK8 regulates a mechanosensitive actin redistribution that maintains immune cell cohesion and protects the nucleus during migration

doi: 10.1101/2024.07.26.605273

Figure Lengend Snippet: Fluorescence intensity of F-actin (phalloidin) in WT or Dock8 KO T cells migrating on fibronectin-coated glass either in absence of (unconfined) or under 1.2% agarose (confined). Representative images (epifluorescence microscope) are shown. (b) Summary plot of F-actin relative fluorescence intensity (RFI) measured at the cell centroid as indicated (top). Data points, individual cells ( n = 166-210 cells); lines, medians. (c, d) Representative WT and Dock8 KO T cells expressing LifeAct-GFP (grey scale, fluorescence intensity) migrating under 1.2% agarose over time acquired by TIRF (c), or widefield microscopy (d). (e, f) Fluorescence intensity of F- actin (phalloidin) in WT and Dock8 KO T cells migrating under 0.5, 1.2 or 2% agarose. Representative examples (epifluorescence microscope) (e), and summary plots (f). Data points, individual cells ( n = 109-140 cells); lines, medians. (g) Two representative example images of F- actin fluorescence intensity in talin1 ( Tln1 )-deficient murine T cells migrating under 1.2% agarose. Statistical tests: Mann-Whitney tests (b); Kruskal-Wallis ANOVA with Dunn’s multiple comparison test (f). *** P < 0.001; ns, non-significant.

Article Snippet: Two-step RT-qPCR: qPCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems) using 2 μL of cDNA per well combined with TaqMan Fast Advanced Master Mix, the TaqMan probe Mm01277042_m1 ( Tbp ) in VIC (endogenous control), and one of the following TaqMan probes, in FAM: Mm00613802_m1 ( Dock8 ), Mm01289583_m1 ( Wwtr1 ) (all reagents from Applied Biosystems).

Techniques: Fluorescence, Microscopy, Expressing, MANN-WHITNEY, Comparison

Journal: bioRxiv

Article Title: DOCK8 regulates a mechanosensitive actin redistribution that maintains immune cell cohesion and protects the nucleus during migration

doi: 10.1101/2024.07.26.605273

Figure Lengend Snippet: Representative images of WT and Dock8 KO T cells migrating under 1.2% agarose. Cells were fixed and stained for F-actin (phalloidin) and nucleus (Hoechst), and images were acquired by epifluorescence microscopy. (b) Histograms of RFI for both F-actin and nucleus from cells shown in (a) taken cross-sectionally across the cell from rear to front. (c) Aggregated histograms of F-actin (phalloidin) and nucleus (Hoechst) RFI of individual T cells ( n = 35 per genotype) normalized over cell length by linear interpolation. Lines for individual cells and means are shown. (d) Representative timelapse images of WT T cells expressing LifeAct-GFP and NLS-nTnG migrating under 1.2% agarose acquired by epifluorescence microscopy. (e) Representative human T cell migrating under 1.2% agarose, fixed and stained for F-actin (phalloidin) and nucleus (Hoechst). (f, g) Representative images (left) and normalized F-actin intensity heatmaps of n = 12-31 cells (right) of WT and Dock 8 KO T cells expressing LifeAct-GFP and NLS-nTnG migrating in straight microchannels with either fixed heights of 5 µm and variable widths of 3, 6 or 8 µm (f), or variable heights of 2.5 or 5 µm and fixed widths of 8 µm (g).

Article Snippet: Two-step RT-qPCR: qPCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems) using 2 μL of cDNA per well combined with TaqMan Fast Advanced Master Mix, the TaqMan probe Mm01277042_m1 ( Tbp ) in VIC (endogenous control), and one of the following TaqMan probes, in FAM: Mm00613802_m1 ( Dock8 ), Mm01289583_m1 ( Wwtr1 ) (all reagents from Applied Biosystems).

Techniques: Staining, Epifluorescence Microscopy, Expressing

Journal: bioRxiv

Article Title: DOCK8 regulates a mechanosensitive actin redistribution that maintains immune cell cohesion and protects the nucleus during migration

doi: 10.1101/2024.07.26.605273

Figure Lengend Snippet: Volcano plots of significant differentially expressed genes (DEG) identified by RNA-seq between WT and Dock8 KO CD8 + T cells activated in vitro and cultured in either media or migrating in collagen (2 mg/ml) for 24 hours with 3 biological replicates per genotype ( FDR < 0.01). FC, fold change. Specific genes of interest (red data points) are labeled. (b) Gene ontology analysis of significant DEGs (445 genes) upregulated in Dock8 KO compared to WT T cells from (a) at FC > 1.5. Bar graph showing gene numbers per category of the top non- redundant enrichment clusters identified with at least 10 genes. Reg., regulation; pos., positive. (c) Heatmap of expression (z-score) of selected genes in WT and Dock8 KO T cells identified among DEGs from (a). (d) Lmna gene expression from RNA-seq data. cpm, counts per million. Data points are independent in vitro activated CD8 + T cell cultures from n = 3 mice per genotype; lines represent medians. (e,f) Lamin A/C expression quantified by flow cytometry in WT or Dock8 KO CD8 + T cells cultured in either media or migrating in collagen (2 mg/ml) for 24 hours. Representative flow cytometry histograms (g), and data summarized from n = 8 mice per genotype and 3 independent experiments (h); lines represent medians; boxes are quartiles; error bars represent the min to max range; RFI are normalized to the mean of WT T cells in media. (g,h) LifeAct-GFP and NLS-nTnG WT and Dock8 KO T cells migrating under 1.2% agarose and imaged using lattice light sheet microscopy. Representative image of a WT T cell shown both from a top view and a side view (g). Summary plot of nuclear height measurements taken from cells that were polarized and not in contact with more than one other cell (h). Data points, n = 22 cells per genotype; lines, means. (i) Alkaline comet assay of WT T cells treated with etoposide (n = 61 cells), or WT (n = 75 cells) and Dock8 KO (n = 52 cells) T cells migrating in collagen (2 mg/ml) for 12 hours. Representative images of visual scoring of double- stranded DNA breaks (score 1 – 4) are shown (right). Statistical tests: one-way ANOVA with Holm-Šídák’s multiple comparisons test (f); Mann-Whitney test (h); * P < 0.05; *** P < 0.001; ns, non-significant.

Article Snippet: Two-step RT-qPCR: qPCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems) using 2 μL of cDNA per well combined with TaqMan Fast Advanced Master Mix, the TaqMan probe Mm01277042_m1 ( Tbp ) in VIC (endogenous control), and one of the following TaqMan probes, in FAM: Mm00613802_m1 ( Dock8 ), Mm01289583_m1 ( Wwtr1 ) (all reagents from Applied Biosystems).

Techniques: RNA Sequencing, In Vitro, Cell Culture, Labeling, Expressing, Gene Expression, Flow Cytometry, Microscopy, Alkaline Single Cell Gel Electrophoresis, MANN-WHITNEY

Journal: bioRxiv

Article Title: DOCK8 regulates a mechanosensitive actin redistribution that maintains immune cell cohesion and protects the nucleus during migration

doi: 10.1101/2024.07.26.605273

Figure Lengend Snippet: Fluorescence intensity of F-actin (phalloidin) in WT, Dock8 KO, or Stk4 KO T cells migrating on fibronectin-coated glass either in absence of (unconfined) or under 1.2% agarose (confined). Representative images (epifluorescence microscope) are shown. (b) Representative confocal microscopy images (max projection of z-stacks) of WT and Stk4 KO T cells migrating through collagen (2 mg/ml). Fixed cells were stained for F-actin (phalloidin), tubulin (α-tubulin antibody), and nucleus (Hoechst). (c) Summary plot of percent WT, Dock8 KO, and Stk4 KO T cells entangled in collagen matrix (2 mg/ml) after 4 hours of migration. Data points, replicate T cell cultures from n = 4 mice per genotype; means are indicated. (d) Summary plot of the frequency of live WT or Dock8 KO T cells either resting in media or after 24 hours of migration in 2mg/ml collagen matrices. Data summarized from n = 3 mice per genotype; frequency is normalized to the mean viability of WT T cells in media. ( e) Lamin A/C expression quantified by flow cytometry in WT, Dock8 KO, or Mst1 KO activated CD8 + T cells migrating in 1.5, 2, or 4 mg/ml collagen for 24 hours. Data summarized from n = 3 mice per genotype; lines, means; RFI is normalized to the mean of WT T cells in 1.5 mg/ml collagen. Dotted line at RFI = 100. (f) Gene expression of Wwtr1 (Taz) assessed by quantitative RT-PCR in WT, Dock 8 KO, or Mst1 KO activated CD8 + T cells cultured in media or migrating in 1.5, 2, or 4 mg/ml collagen for 24 hours. Expression was normalized to housekeeping gene Tbp and shown as fold change compared to WT T cells in media. Dotted line at fold = 1; data summarized from n = 3-6 mice per genotype from 1-2 independent experiment; lines, means. (f) Gene expression of Dock8 assessed by quantitative RT-PCR in WT or Mst1 KO activated CD8 + T cells cultured in media or migrating in 1.5, 2, or 4 mg/ml collagen for 24 hours. Expression was normalized to a housekeeping gene and shown as fold change compared to the WT T cells in media. Dotted line at fold = 1; data summarized from n = 3 mice per genotype from 1 experiment; lines, means.

Article Snippet: Two-step RT-qPCR: qPCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems) using 2 μL of cDNA per well combined with TaqMan Fast Advanced Master Mix, the TaqMan probe Mm01277042_m1 ( Tbp ) in VIC (endogenous control), and one of the following TaqMan probes, in FAM: Mm00613802_m1 ( Dock8 ), Mm01289583_m1 ( Wwtr1 ) (all reagents from Applied Biosystems).

Techniques: Fluorescence, Microscopy, Confocal Microscopy, Staining, Migration, Expressing, Flow Cytometry, Gene Expression, Quantitative RT-PCR, Cell Culture

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: Frontiers in Immunology

Article Title: Deletion of Dock10 in B Cells Results in Normal Development but a Mild Deficiency upon In Vivo and In Vitro Stimulations

doi: 10.3389/fimmu.2017.00491

Figure Lengend Snippet: Interleukin-4 (IL-4) induces upregulation of Dock10 expression in murine primary B cells and malignant B cell lines . (A–D,H,I) mRNA expression of various genes by real-time reverse transcriptase polymerase chain reaction. The values represent the mean and SD of three biological replicates. (A–D) Values are compared to the reference gene Mb-1, which is set to 1. (H,I) Values are compared to the reference gene GAPDH, which is set to 1. (E–G) Protein expression as analyzed by Western blot. The relative intensities are specified relative to that of α-tubulin. (A,B) mRNA expression of (A) Dock10 and Dock 11 and (B,C) Dock8 and Dock10 after 24 h stimulation of spleen B cells. Values are shown as fold increase normalized to non-stimulated spleen B cells. (D) Kinetics of Dock10 mRNA after IL-4 stimulation. Cells were harvested at the indicated time points. (E) Dock10 protein expression in spleen B cells upon stimulation for 24 h. (F) Kinetics of Dock10 protein expression in spleen B cells stimulated for 0–46 h with IL-4. (G) Dock10 protein expression in spleen B cells after stimulation for 24 or 48 h. (H) Dock10 expression in mouse B cell lines. The results are shown as fold expression of Dock10, normalized to the pre-B cell line 70Z/3 set to 1. (I) Fold increase of Dock10 expression in mouse B cells incubated for 24 h with IL-4. The data are representative of three experiments (A–D) , four experiments (E) , and two experiments (F–I) with similar results.

Article Snippet: RT-PCR was carried out using TaqMan Master Mix and 20× TaqMan Gene Expression Assays [Dock8: Mm00472344_m1, Dock10: Mm00614273_m1, Dock11: Mm01297590_m1, GAPDH: Mm03302249_m1, and Mb-1 (CD79a): Mm00432423_m1, Applied Biosystems].

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Incubation

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

Journal: Clinical Immunology (Orlando, Fla.)

Article Title: Hypomorphic function and somatic reversion of DOCK8 cause combined immunodeficiency without hyper-IgE

doi: 10.1016/j.clim.2015.12.003

Figure Lengend Snippet: A novel compound heterozygous mutation in DOCK8 results in expression of a truncated DOCK8 protein. (A) Sanger sequencing results for the single nucleotide duplication, c.6019dupT, p.(Tyr2007Leufs*12). The upper panel illustrates a normal control trace and the lower panel shows the presence of the mutation; the duplicated T nucleotide is indicated by the arrow. (B) Results of array comparative genomic hybridization illustrating the about 140 kb deletion in 9p24.3 (204,193–343,954). The deletion encompasses exons 1–14 of DOCK8 . (C) Graphic depicting the wild-type DOCK8 protein structure and the outcome of the single-nucleotide insertion on the maternal allele and the deletion in DOCK8 on the paternal allele on DOCK8 protein expression ( DOCK8 transcript reference is ENST00000453981). (D) DOCK8 protein expression in EBV-transformed B cells of a healthy control (7.5 μg protein lysate) and the patient (30 μg protein lysate). Actin was used as loading control.

Article Snippet: Somatic reversion of DOCK8 in T cells was confirmed by pyrosequencing of DOCK8 ( D).

Techniques: Mutagenesis, Expressing, Sequencing, Control, Hybridization, Transformation Assay

Journal: Clinical Immunology (Orlando, Fla.)

Article Title: Hypomorphic function and somatic reversion of DOCK8 cause combined immunodeficiency without hyper-IgE

doi: 10.1016/j.clim.2015.12.003

Figure Lengend Snippet: Improvement of T cell proliferation over time, somatic reversion of DOCK8 in T cells and hypomorphic function of the truncated DOCK8 protein. Proliferation of PHA-stimulated, CFSE-labeled PBMCs of the patient at (A) 10 and (B) 15 years of age, and a healthy control. Depicted are percentages of CFSE low cells gated on CD3 + CD4 + or CD4 − or CD8 + T cells. (C) Sanger sequence trace showing somatic reversion of the single nucleotide duplication (c.6019dupT) resulting in expression of about 60% wild-type DOCK8 transcripts in the patient's CD3 + T cells. (D) “T” nucleotide phosphorescence ratios obtained by pyrosequencing DOCK8 of primary CD3 + CD4 + and CD3 + CD8 + T cells, primary CD19 + B cells and the EBV B cell line of the patient. (T/± 1 or 2) depicts the signal ratio of c.6018-19T to nucleotides 1 and 2 positions up and downstream. The PCR templates and pyrosequencing reactions were performed in triplicate. Each symbol represents the mean of the three ratio measurements at respective nucleotide positions. The bar represents the mean “T” nucleotide phosphorescence ratio of all 4 different nucleotide ratios in indicated cell populations. (E) Sanger sequence trace showing expression of solely mutated DOCK8 transcripts in EBV-transformed B cells of the patient. The duplicated T-nucleotide is indicated by the arrow and #. (F) Migration of EBV-transformed B cells of 5 different healthy controls (each symbol represents the mean of 3 independent experiments for each of the healthy control samples), the patient and a patient with a complete loss-of-function mutation in DOCK8 (DOCK8 null ). The bar of the healthy control samples represents the mean of the mean of each of the 5 healthy control samples. The bar for each of the patient samples represents mean and standard deviation of 3 independent experiments for each sample.

Article Snippet: Somatic reversion of DOCK8 in T cells was confirmed by pyrosequencing of DOCK8 ( D).

Techniques: Labeling, Control, Sequencing, Expressing, Transformation Assay, Migration, Mutagenesis, Standard Deviation