Journal: Nature Communications

Article Title: Cancer immune control needs senescence induction by interferon-dependent cell cycle regulator pathways in tumours

doi: 10.1038/s41467-020-14987-6

Figure Lengend Snippet: a Individual follow-up of tumour volumes. CD8-depleted mice were subcutaneously (s.c.) engrafted with 1 × 10 6 RT2-, RT2. Stat1 −/− -, RT2. Cdkn2a −/− - or RT2.CRISPR- Cdkn2a -cancer cells. Treatment with isotype control mAbs (Ctr) or combined immune checkpoint inhibitors (ICB; anti-PD-L1 and anti-LAG-3) once per week was started when tumours were >3 mm in diameter. Cancer size was measured 2 times per week. Number of mice with regressing tumours and the total number of mice is given in parenthesis; RT2 Ctr N = 9, ICB N = 10, RT2. Stat1 −/− Ctr N = 6, ICB N = 8, RT2. Cdkn2a −/− Ctr N = 12, ICB N = 14, RT2.CRISPR- Cdkn2a Ctr N = 5, ICB N = 10. Each cell line was given a different lining. Black lines summarise the results for different treatment groups (as obtained from ANCOVA). p -values examine the question whether the treatment effect was different between two genotypes. Mice were killed either when tumours reached the critical diameter of 15–20 mm or ulcerated, or when mice developed signs of wasting. b Representative triple-staining for the senescence marker p16 Ink4a (red) and the proliferation marker Ki67 (blue) and for nuclei (green) of the s.c. tumour of individual mice treated as described in ( a ). Scale bar 2 µm. c Individual data points showing quantification of p16 Ink4a+ (left) or Ki67 + (right) cells. Each data point represents the total of three tumour slides measurements, tumours of three individual mice (described in a ) were analysed. In a significance was tested by using unequal variances t -test, p -values examines the treatment effect, comparing the ICB-treated RT2-cancers with each ICB-treated knock-out group.

Article Snippet: Cdkn2a −/− - or RT2.CRISPR- Cdkn2a -cancer cells were s.c. transplanted, three days after depletion of CD8 cells with 100 μg anti-CD8 mAb (Rm-CD8-2 AK, Core Facility mAb, Helmholtz-Zentrum München).

Techniques: CRISPR, Control, Staining, Marker, Knock-Out

Journal: Nature Communications

Article Title: Cellular census of human fibrosis defines functionally distinct stromal cell types and states

doi: 10.1038/s41467-020-16264-y

Figure Lengend Snippet: a Force directed graph (Fruchterman–Reingold layout) of single-cell RNA-seq showing myofibroblasts coloured by major subsets ( n = 12 DD patients). MFB represents myofibroblast. b Heatmap of single cell RNA-seq showing the z -score expression of top ten myofibroblast subset markers. Two-sided Wilcoxon Rank Sum Test with FDR correction (BH correction ( n = 12 DD patients). c tSNE projections of single cell RNA-seq showing myofibroblasts coloured by the expression of ACTA2 and MKI67 in scaled log(UMI + 1) ( n = 12 DD patients). d Violin plots showing gene expression of MKI67 and ITGB1 in fibroblasts and myofibroblasts in scaled(log(UMI + 1)) from single cell RNA-seq. e Box and whisker plot of flow cytometry analysis showing Ki67 protein expression in myofibroblasts ITGβ1 high myofibroblasts (range 21–46%, mean 28% and box bounds 24–27% representing first to third quantiles) and ITGβ1 low fibroblasts (range 0–0%, mean 0.0% and percentiles 0%). Two-sided unpaired t test, p value = 0.00014, mean ± SEM. ( n = 8 DD patients). f Dot plot of single cell RNA-seq showing pathways enriched in myofibroblast subsets. Gene ratio is number of marker genes associated with pathway and p .adjust is adjusted p value (two-sided Wilicoxon Rank Sum test, BH FDR-correction). g tSNE projections of CyTOF analysis for representative DD patient showing distinct CD82 high OX40L + myofibroblast. Scale bar is normalised protein expression. ( n = 6 DD patients). h Confocal images of immunofluorescence showing co-expression of CD82, α-SMA and ITG-β1 in DD nodules ( n = 3 DD patients). Scale bar 20 µm.

Article Snippet: CD252 169-Tm (3166007B, ML5, Fluidigm), CD34 166-Er (Catalog:3166012B, Clone:581, Fluidigm), CD45 141-Pr (Catalog: 3141009B, Clone: HI30, Fluidigm), HLAD A, B C 142-Nd (Catalog: 3142007B, Clone: HCD57, Fluidigm), CD19 143-Nd (Catalog: 3144007 A, Clone:NP6G4, Fluidigm), HLA-DR 89-Y (Catalog: 3173005B, L243, Fluidigm), CD3 189-Y (Catalog: 3158021 A, Clone: 24E10, Fluidigm), B-catenin 176-Lu (Catalog: 3147005 A, Clone:D10A8, Fluidigm), CD55 174-Yb (Catalogue; 3148015B, Clone: JS11, Fluidigm), CD146 155-Nd (3155006B, P1H12, Fluidigm), CD29 156-Gd (3156007B, TS2/16, Fluidigm), CD82 158Gd (3158025B, ASL-24, Fluidigm), CD90 172-Yb (3173011B, 5E10, Fluidigm), TGFb 150-Nd (3163010B, CloneTW46H10, Fluidigm) CD163 145-Nd (3145010B, GHI/61, Fluidigm), Ki-67 168-Er (3168007B, B56, Fluidigm), CD54 170-Er (3170014B, HA58, Fluidigm), CD68 171-Yb (3171011B, Y1/82A, Fluidigm), CD9 172-Yb (Cat. No. 312102, Biolegend), CD252 150-Nd (326302, Biolegend), PGLA2 172-Yb (MAB5374, R&D Systems), a-SMA 156-Gr (MAB1420, 1A4, R&D Systems), gp38 155-Gd (AF3670-SP, R&D Systems), PDGFRB 154-Sm (SAB4700458, 18A2, Sigma Aldrich), MMP14 149-Sm (MAB9181-SP, 128527 R&D Systems), Cad-11 148-Nd (AF1790-SP, R&D Systems) and FAP 147-Sm (MAB3715-SP, 427819 RD Systems).

Techniques: RNA Sequencing, Expressing, Gene Expression, Whisker Assay, Flow Cytometry, Marker, Immunofluorescence

Journal: Nature Communications

Article Title: Cellular census of human fibrosis defines functionally distinct stromal cell types and states

doi: 10.1038/s41467-020-16264-y

Figure Lengend Snippet: a Line plot demonstrating CD82 expression in myofibroblasts ( loess smoothed normalised counts ± SE) along pseudotime (myofibroblast activation trajectory) in single-cell RNA-seq ( n = 12 DD patients). b Above, Heatmaps of traction force microscopy analysis showing magnitude of traction force (Pascals) in freshly isolated FACS sorted myofibroblasts (CD82 high and CD82 low ). Below, surface plots of traction force microscopy showing representative force foci from sorted myofibroblasts (Scaled to 800 Pa). Colour scale represents force range per cell. c Bar plots of traction force microscopy analysis showing mean and maximum traction force per cell in CD82 high and CD82 low sorted myofibroblast populations ( n = 3 DD patients, >15 cells per condition). Two-sided unpaired t test, mean ± SEM. p Values = 0.0056 (max traction), and 0.0098 (mean traction). d tSNE projection of traction force microscopy analysis showing clustering of single cell force signatures in sorted populations (CD82 high and CD82 low ) ( n = 3 DD patients, >15 cells per condition). e Box and whisker plot of flow cytometry analysis showing cell cycle phases in siControl and siCD82 transfected DD myofibroblasts ( n = 3 DD patients). Two-sided paired t test, p value = 0.023, mean ± SEM). (siRNA range 2.6–85.5%, mean 32.8% and box bounds 9.75–45.2% represent first and third quantiles. siCD82 range 1.1–87.1%, mean 32.7% and box bounds 3.1–59.8% represent first and third quantiles.) f tSNE projections of single-cell RNA-seq showing fibroblasts and myofibroblasts in bleomycin murine model marked by the expression of selected genes in scaled log(UMI + 1) ( n = 2 mice). g tSNE projection of single cell RNA-seq showing murine fibroblasts and myofibroblasts in bleomycin model coloured by Louvain clusters ( n = 2 mice). h Bar plot of single cell RNA-seq showing the percentage of stromal cells before (Day 0) and after (Day 21) the installation of bleomycin in murine lung fibrosis model. i Confocal images of immunofluorescence showing co-expression of CD82, α-SMA and PDGFR-α in human IPF ( n = 3 independent IPF patients). Scale bar = 20 µm.

Article Snippet: CD252 169-Tm (3166007B, ML5, Fluidigm), CD34 166-Er (Catalog:3166012B, Clone:581, Fluidigm), CD45 141-Pr (Catalog: 3141009B, Clone: HI30, Fluidigm), HLAD A, B C 142-Nd (Catalog: 3142007B, Clone: HCD57, Fluidigm), CD19 143-Nd (Catalog: 3144007 A, Clone:NP6G4, Fluidigm), HLA-DR 89-Y (Catalog: 3173005B, L243, Fluidigm), CD3 189-Y (Catalog: 3158021 A, Clone: 24E10, Fluidigm), B-catenin 176-Lu (Catalog: 3147005 A, Clone:D10A8, Fluidigm), CD55 174-Yb (Catalogue; 3148015B, Clone: JS11, Fluidigm), CD146 155-Nd (3155006B, P1H12, Fluidigm), CD29 156-Gd (3156007B, TS2/16, Fluidigm), CD82 158Gd (3158025B, ASL-24, Fluidigm), CD90 172-Yb (3173011B, 5E10, Fluidigm), TGFb 150-Nd (3163010B, CloneTW46H10, Fluidigm) CD163 145-Nd (3145010B, GHI/61, Fluidigm), Ki-67 168-Er (3168007B, B56, Fluidigm), CD54 170-Er (3170014B, HA58, Fluidigm), CD68 171-Yb (3171011B, Y1/82A, Fluidigm), CD9 172-Yb (Cat. No. 312102, Biolegend), CD252 150-Nd (326302, Biolegend), PGLA2 172-Yb (MAB5374, R&D Systems), a-SMA 156-Gr (MAB1420, 1A4, R&D Systems), gp38 155-Gd (AF3670-SP, R&D Systems), PDGFRB 154-Sm (SAB4700458, 18A2, Sigma Aldrich), MMP14 149-Sm (MAB9181-SP, 128527 R&D Systems), Cad-11 148-Nd (AF1790-SP, R&D Systems) and FAP 147-Sm (MAB3715-SP, 427819 RD Systems).

Techniques: Expressing, Activation Assay, RNA Sequencing, Microscopy, Isolation, Whisker Assay, Flow Cytometry, Transfection, Immunofluorescence

Journal: Immunotherapy

Article Title: α-PD-L1 mAb enhances the abscopal effect of hypo-fractionated radiation by attenuating PD-L1 expression and inducing CD8 + T-cell infiltration.

doi: 10.2217/imt-2018-0049

Figure Lengend Snippet: Figure 1. Cooperativity of immunoradiation in a metastatic lung cancer model. (A) All of the mice (including mice receiving mock radiation) were lightly anesthetized via the i.p. route. RT was administered locally and exclusively to the irradiated tumor, and the rest of the body was shielded. A single dose or multiple fractions were administered on consecutive days starting on day 13. The PD-L1-blocking mAb 9H10 was given i.p. four-times every 3 days, at days 13, 16, 19 and 22. Tumor volumes were measured every other day until day 30, when the mice were sacrificed. All treatments were primed on day 13. (B–E) Tumor growth curve (left panel) and tumor volume at day 10 after treatment (day 13 after tumor inoculation, right panel). (B) Irradiated tumor. (C) Nonirradiated tumor. (D & E) Depletion of CD4+ or CD8+ T cells in the 8 Gy × 3 F + α-PD-L1 group. (D) Irradiated tumor. (E) Nonirradiated tumor. The data are the means ± SEM of four to six mice per group. *p < 0.05; **p < 0.01; ***p < 0.001. i.p.: Intraperitoneal; mAb: Monoclonal antibody; PD-L1: Programmed death ligand 1; RT: Radiotherapy; SEM: Standard error of the mean.

Article Snippet: To achieve T-cell depletion, 250 mg of monoclonal α-CD4 (GK1.5 clone, BioXCell #BE003-1) or α-CD8 (2.43 clone, BioXCell #BE0061) antibodies were intraperitoneally administered, and depletion was verified by peripheral blood mononuclear cell staining (Supplementary Figure 3).

Techniques: Irradiation, Blocking Assay

Journal: Immunotherapy

Article Title: α-PD-L1 mAb enhances the abscopal effect of hypo-fractionated radiation by attenuating PD-L1 expression and inducing CD8 + T-cell infiltration.

doi: 10.2217/imt-2018-0049

Figure Lengend Snippet: Figure 1. Cooperativity of immunoradiation in a metastatic lung cancer model (cont.). (A) All of the mice (including mice receiving mock radiation) were lightly anesthetized via the i.p. route. RT was administered locally and exclusively to the irradiated tumor, and the rest of the body was shielded. A single dose or multiple fractions were administered on consecutive days starting on day 13. The PD-L1-blocking mAb 9H10 was given i.p. four-times every 3 days, at days 13, 16, 19 and 22. Tumor volumes were measured every other day until day 30, when the mice were sacrificed. All treatments were primed on day 13. (B–E) Tumor growth curve (left panel) and tumor volume at day 10 after treatment (day 13 after tumor inoculation, right panel). (B) Irradiated tumor. (C) Nonirradiated tumor. (D & E) Depletion of CD4+ or CD8+ T cells in the 8 Gy × 3 F + α-PD-L1 group. (D) Irradiated tumor. (E) Nonirradiated tumor. The data are the means ± SEM of four to six mice per group. *p < 0.05; **p < 0.01; ***p < 0.001. i.p.: Intraperitoneal; mAb: Monoclonal antibody; PD-L1: Programmed death ligand 1; RT: Radiotherapy; SEM: Standard error of the mean.

Article Snippet: To achieve T-cell depletion, 250 mg of monoclonal α-CD4 (GK1.5 clone, BioXCell #BE003-1) or α-CD8 (2.43 clone, BioXCell #BE0061) antibodies were intraperitoneally administered, and depletion was verified by peripheral blood mononuclear cell staining (Supplementary Figure 3).

Techniques: Irradiation, Blocking Assay

Journal: Immunotherapy

Article Title: α-PD-L1 mAb enhances the abscopal effect of hypo-fractionated radiation by attenuating PD-L1 expression and inducing CD8 + T-cell infiltration.

doi: 10.2217/imt-2018-0049

Figure Lengend Snippet: Figure 1. Cooperativity of immunoradiation in a metastatic lung cancer model (cont.). (A) All of the mice (including mice receiving mock radiation) were lightly anesthetized via the i.p. route. RT was administered locally and exclusively to the irradiated tumor, and the rest of the body was shielded. A single dose or multiple fractions were administered on consecutive days starting on day 13. The PD-L1-blocking mAb 9H10 was given i.p. four-times every 3 days, at days 13, 16, 19 and 22. Tumor volumes were measured every other day until day 30, when the mice were sacrificed. All treatments were primed on day 13. (B–E) Tumor growth curve (left panel) and tumor volume at day 10 after treatment (day 13 after tumor inoculation, right panel). (B) Irradiated tumor. (C) Nonirradiated tumor. (D & E) Depletion of CD4+ or CD8+ T cells in the 8 Gy × 3 F + α-PD-L1 group. (D) Irradiated tumor. (E) Nonirradiated tumor. The data are the means ± SEM of four to six mice per group. *p < 0.05; **p < 0.01; ***p < 0.001. i.p.: Intraperitoneal; mAb: Monoclonal antibody; PD-L1: Programmed death ligand 1; RT: Radiotherapy; SEM: Standard error of the mean.

Article Snippet: To achieve T-cell depletion, 250 mg of monoclonal α-CD4 (GK1.5 clone, BioXCell #BE003-1) or α-CD8 (2.43 clone, BioXCell #BE0061) antibodies were intraperitoneally administered, and depletion was verified by peripheral blood mononuclear cell staining (Supplementary Figure 3).

Techniques: Irradiation, Blocking Assay

Journal: Immunotherapy

Article Title: α-PD-L1 mAb enhances the abscopal effect of hypo-fractionated radiation by attenuating PD-L1 expression and inducing CD8 + T-cell infiltration.

doi: 10.2217/imt-2018-0049

Figure Lengend Snippet: Figure 2. Radiotherapy-induced tumor-infiltrating T cells in the tumor environment showed a combined effect with α-programmed death ligand 1. At days 5 and 10 after RT (day 13 after tumor inoculation), the mice were sacrificed, and the tumor cells were analyzed. The data are representative of at least two independent experiments (n > 3). (A & B) CD45+ cells among lymphocytes at days 5 and 10 after RT. (A) Irradiated tumor. (B) Nonirradiated tumor (right panel). (C & D) Percentage of CD8+ T cells within the CD45+ cell population. (C) Irradiated tumor. (D) Nonirradiated tumor. (E & F) Percentage of MDSCs (CD11b+ Gr1+) in the population of CD45+ cells. (E) Irradiated tumor. (F) Nonirradiated tumor. *p < 0.05; **p < 0.01; ***p < 0.001. RT: Radiotherapy; MDSC: Myeloid-derived suppressor cell.

Article Snippet: To achieve T-cell depletion, 250 mg of monoclonal α-CD4 (GK1.5 clone, BioXCell #BE003-1) or α-CD8 (2.43 clone, BioXCell #BE0061) antibodies were intraperitoneally administered, and depletion was verified by peripheral blood mononuclear cell staining (Supplementary Figure 3).

Techniques: Irradiation, Derivative Assay

Journal: Immunotherapy

Article Title: α-PD-L1 mAb enhances the abscopal effect of hypo-fractionated radiation by attenuating PD-L1 expression and inducing CD8 + T-cell infiltration.

doi: 10.2217/imt-2018-0049

Figure Lengend Snippet: Figure 2. Radiotherapy-induced tumor-infiltrating T cells in the tumor environment showed a combined effect with α-programmed death ligand 1 (cont.). At days 5 and 10 after RT (day 13 after tumor inoculation), the mice were sacrificed, and the tumor cells were analyzed. The data are representative of at least two independent experiments (n > 3). (A & B) CD45+ cells among lymphocytes at days 5 and 10 after RT. (A) Irradiated tumor. (B) Nonirradiated tumor (right panel). (C & D) Percentage of CD8+ T cells within the CD45+ cell population. (C) Irradiated tumor. (D) Nonirradiated tumor. (E & F) Percentage of MDSCs (CD11b+ Gr1+) in the population of CD45+ cells. (E) Irradiated tumor. (F) Nonirradiated tumor. *p < 0.05; **p < 0.01; ***p < 0.001. RT: Radiotherapy; MDSC: Myeloid-derived suppressor cell.

Article Snippet: To achieve T-cell depletion, 250 mg of monoclonal α-CD4 (GK1.5 clone, BioXCell #BE003-1) or α-CD8 (2.43 clone, BioXCell #BE0061) antibodies were intraperitoneally administered, and depletion was verified by peripheral blood mononuclear cell staining (Supplementary Figure 3).

Techniques: Irradiation, Derivative Assay

Journal: Immunotherapy

Article Title: α-PD-L1 mAb enhances the abscopal effect of hypo-fractionated radiation by attenuating PD-L1 expression and inducing CD8 + T-cell infiltration.

doi: 10.2217/imt-2018-0049

Figure Lengend Snippet: Figure 4. Radiotherapy-induced programmed death ligand 1 expression in the microenvironment can be downregulated by α-programmed death ligand 1. At days 5 and 10 after the first treatment was administered (at day 13 after tumor inoculation), the mice were sacrificed, and the tumor cells were analyzed. The data are representative of two or three independent experiments (n > 3). The control samples were obtained from untreated mice with a time-matched sample. (A–C) PD-L1 expression in tumors: (A) irradiated tumor, (B) nonirradiated tumor and (C) histogram comparing PD-L1 expression in the three groups. (D & E) PD1 expression on intratumoural CD8+ T cells: (D) irradiated tumor and (E) nonirradiated tumor. (F & G) PD-L1 expression on intratumoural CD11b+ Gr1+ cells: (F) irradiated tumor and (G) nonirradiated tumor. *p < 0.05; **p < 0.01; ***p < 0.001. PD-L1: Programmed death ligand 1.

Article Snippet: To achieve T-cell depletion, 250 mg of monoclonal α-CD4 (GK1.5 clone, BioXCell #BE003-1) or α-CD8 (2.43 clone, BioXCell #BE0061) antibodies were intraperitoneally administered, and depletion was verified by peripheral blood mononuclear cell staining (Supplementary Figure 3).

Techniques: Expressing, Control, Irradiation

Journal: Immunotherapy

Article Title: α-PD-L1 mAb enhances the abscopal effect of hypo-fractionated radiation by attenuating PD-L1 expression and inducing CD8 + T-cell infiltration.

doi: 10.2217/imt-2018-0049

Figure Lengend Snippet: Figure 4. Radiotherapy-induced programmed death ligand 1 expression in the microenvironment can be downregulated by α-programmed death ligand 1 (cont.). At days 5 and 10 after the first treatment was administered (at day 13 after tumor inoculation), the mice were sacrificed, and the tumor cells were analyzed. The data are representative of two or three independent experiments (n > 3). The control samples were obtained from untreated mice with a time-matched sample. (A–C) PD-L1 expression in tumors: (A) irradiated tumor, (B) nonirradiated tumor and (C) histogram comparing PD-L1 expression in the three groups. (D & E) PD1 expression on intratumoural CD8+ T cells: (D) irradiated tumor and (E) nonirradiated tumor. (F & G) PD-L1 expression on intratumoural CD11b+ Gr1+

Article Snippet: To achieve T-cell depletion, 250 mg of monoclonal α-CD4 (GK1.5 clone, BioXCell #BE003-1) or α-CD8 (2.43 clone, BioXCell #BE0061) antibodies were intraperitoneally administered, and depletion was verified by peripheral blood mononuclear cell staining (Supplementary Figure 3).

Techniques: Expressing, Control, Irradiation

Journal: Cells

Article Title: Long-Term Engraftment and Satellite Cell Expansion from Human PSC Teratoma-Derived Myogenic Progenitors

doi: 10.3390/cells14151150

Figure Lengend Snippet: Scheme outlining the key steps of the study. hiPSCs were injected into TA muscles of NSG mice, and teratomas were harvested two months later. Tissue was digested, and skeletal myogenic progenitor cells co-expressing CD82, ERBB3, and NGFR were sorted by FACS, cultured in vitro, tested for differentiation into myotubes, and, in parallel, transplanted into tibialis anterior (TA) muscles of injured NSG-mdx 4Cv mice following X-ray and cardiotoxin injection. TA muscle tissue was harvested at various time points, and stained sections were analyzed with different antibodies to assess human cell contribution, muscle regeneration, and fiber type differentiation.

Article Snippet: The dissociated cells were incubated with antibodies APC anti-CD82 (FAB4616A, R&D Systems, Minneapolis, MN, USA; RRID: AB_2076404), PE anti-ERBB3 (304706, BioLegend, San Diego, CA, USA; RRID: AB_2099569), and PECy7 anti-NGFR (Cat# 562122, BD Biosciences, San Jose, CA, USA; RRID: AB_10894762); each at 0.5 uL per 1 million cells) in FACS buffer for one hour on ice.

Techniques: Injection, Muscles, Expressing, Cell Culture, In Vitro, Staining

Journal: Cells

Article Title: Long-Term Engraftment and Satellite Cell Expansion from Human PSC Teratoma-Derived Myogenic Progenitors

doi: 10.3390/cells14151150

Figure Lengend Snippet: ( A ) Morphology of teratomas formed in TA muscles of NSG mice two months after injection of hiPSCs. ( B ) Isolation of skeletal myogenic progenitor cells co-expressing CD82, ERBB3, and NGFR by FACS from TA teratomas. ( C ) Expression levels of various myogenic and fibroblastic markers under different growth factor conditions, as determined by RNA sequencing, presenting mean ratio normalized counts. ( D ) Morphology of isolated skeletal myogenic cells cultured at different passages. PAX7 staining of cells in the proliferative phase at confluency, from the same passage used for transplantation. ( E ) MyoD, Myogenin, MF-20, and PAX7 staining of teratoma-derived skeletal myogenic cells in differentiation medium at passage 3, highlighting their capacity to form myotubes. ( F ) Percentage of cells positive for PAX7 in proliferative and differentiation media, and for MyoD, Myogenin, and MF-20 in differentiation medium. Data were obtained from five fields per sample across three replicates.

Article Snippet: The dissociated cells were incubated with antibodies APC anti-CD82 (FAB4616A, R&D Systems, Minneapolis, MN, USA; RRID: AB_2076404), PE anti-ERBB3 (304706, BioLegend, San Diego, CA, USA; RRID: AB_2099569), and PECy7 anti-NGFR (Cat# 562122, BD Biosciences, San Jose, CA, USA; RRID: AB_10894762); each at 0.5 uL per 1 million cells) in FACS buffer for one hour on ice.

Techniques: Muscles, Injection, Isolation, Expressing, RNA Sequencing, Cell Culture, Staining, Transplantation Assay, Derivative Assay

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: αβ T-cell receptors from multiple sclerosis brain lesions show MAIT cell–related features

doi: 10.1212/NXI.0000000000000107

Figure Lengend Snippet: (A) Cluster of expanded Vβ1 + T cells within multiple sclerosis (MS) lesions. Immunohistochemistry for the expanded and persisting Vβ1 clone (green) in clusters of CD8 + T cells (red) in parenchymal MS lesions. Several such clusters were observed. Only very few scattered CD8 − Vβ1 + T cells could be identified in the brain lesion. Nuclei are visualized with 4',6-diamidino-2-phenylindole (white). Scale bar 20 µm. (B) Sequences of paired T-cell receptor (TCR) α and β chains. Single sorted or laser microdissected Vβ1 + CD8 + T cells from peripheral blood or brain sections were submitted to single-cell TCR PCR to identify Vβ1 chains and all possible matching α chains. The V, n(D)n, and J regions are indicated. Amino acids encoded by n(D)n nucleotides are printed in red. The expanded Vβ1-Jβ2.3 β chain (upper line) was found to pair with 4 different α chains. Three α chains were identified from brain lesions, and 1 α chain was found in blood. The α chains expressing the Jα33 (second line) and Jα16 (fifth line) elements were identified in 7 and 6 independent cells, respectively. All α chains share the Vα7.2 element, and even though they do not share the same Jα element, they all show homologous complementarity determining region 3α regions with a conserved valine (V) followed by a positively charged arginine (R) (with only one clone showing a glutamine [Q]), a negatively charged amino acid (D/E), and a relatively large hydrophilic amino acid. One of the α chains (highlighted in red) is the mucosal-associated invariant T (MAIT) cell canonical TCR Vα7.2-CAXXDSNYQLIW-Jα33 chain with 2 N nucleotide–encoded amino acids between Vα7.2 and Jα33 (here VR). The other clones with Jα16, Jα24.1, and Jα58 chains are atypical for MAIT cells, which usually carry Jα33, Jα20, or Jα12. PBMC = peripheral blood mononuclear cell.

Article Snippet: To characterize T-cell infiltrates in sections of MS brain, the following antibodies against cell surface molecules were used: mouse anti-human CD161 (1:5, 191B8, Miltenyi Biotec, Bergisch Gladbach, Germany), mouse anti-human Vα7.2 (1:5, 3C10, BioLegend, San Diego, CA), mouse anti-human CD8α (1:50, LT8, AbD Serotec, Kidlington, UK; labeled with the Cy3 MAb labeling kit, GE Healthcare, Freiburg, Germany), rabbit anti-human CCR7 (1:800, Y59, Abcam, Cambridge, UK), mouse anti-human CD45RA (1:250, HI100, BioLegend), mouse anti-human CD45RO (1:250, UCHL1, BioLegend), fluorescein isothiocyanate (FITC)-labeled mouse anti-human Vβ1 (1:100, BL37.2, Beckman Coulter, Brea, CA), rabbit anti-human CD3 (1:500, Dako, Glostrup, Denmark), and Alexa Fluor 488–conjugated mouse anti-human CD4 (1:50, RPA-T4, eBioscience, San Diego, CA).

Techniques: Immunohistochemistry, Expressing, Clone Assay

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: αβ T-cell receptors from multiple sclerosis brain lesions show MAIT cell–related features

doi: 10.1212/NXI.0000000000000107

Figure Lengend Snippet: Immunofluorescence staining of brain-infiltrating immune cells. All nuclei are stained with 4′,6-diamidino-2-phenylindole (white). Green and red dyes were used. Double-positive cells are therefore shown in yellow. Scale bars 50 µm. (A) Double staining for CD3 (green) and CD8α (red). Most CD3 + T cells coexpress CD8α. (B) CD8 + (red) T cells outnumber CD4 + (green) T cells. (C) Low numbers of CD45RA + (green) CD8 + (red) double-positive T cells in multiple sclerosis (MS) brain tissue. (D) Many CD8 + T cells (red) coexpress CD45RO (green). (E) Naive T cells double-positive for CCR7 (red) and CD45RA (green) are mostly found within blood vessels and are barely detectable in the parenchyma of MS CNS. (F) Effector memory (CD45RO + CCR7 − ) (green arrow) and central memory (CD45RO + CCR + ) (yellow arrow) T cells in MS lesions.

Article Snippet: To characterize T-cell infiltrates in sections of MS brain, the following antibodies against cell surface molecules were used: mouse anti-human CD161 (1:5, 191B8, Miltenyi Biotec, Bergisch Gladbach, Germany), mouse anti-human Vα7.2 (1:5, 3C10, BioLegend, San Diego, CA), mouse anti-human CD8α (1:50, LT8, AbD Serotec, Kidlington, UK; labeled with the Cy3 MAb labeling kit, GE Healthcare, Freiburg, Germany), rabbit anti-human CCR7 (1:800, Y59, Abcam, Cambridge, UK), mouse anti-human CD45RA (1:250, HI100, BioLegend), mouse anti-human CD45RO (1:250, UCHL1, BioLegend), fluorescein isothiocyanate (FITC)-labeled mouse anti-human Vβ1 (1:100, BL37.2, Beckman Coulter, Brea, CA), rabbit anti-human CD3 (1:500, Dako, Glostrup, Denmark), and Alexa Fluor 488–conjugated mouse anti-human CD4 (1:50, RPA-T4, eBioscience, San Diego, CA).

Techniques: Immunofluorescence, Staining, Double Staining

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: αβ T-cell receptors from multiple sclerosis brain lesions show MAIT cell–related features

doi: 10.1212/NXI.0000000000000107

Figure Lengend Snippet: Double fluorescence immunohistochemistry identifies brain-infiltrating mucosal-associated invariant T (MAIT) cells in multiple sclerosis (MS) lesions of patient A. Nuclei are stained with 4',6-diamidino-2-phenylindole (white). Green and red dyes were used. Double-positive cells are therefore shown in yellow. Scale bars 20 µm. (A) T cells expressing the T-cell receptor Vα7.2 (red) and Vβ1 chains (green). This combination (see ) was identified by single-cell PCR. (B) Most Vα7.2 + (green) T cells belong to the CD8 + (red) T-cell subset. (C) MAIT cells expressing Vα7.2 (red) and CD161 (green) in the parenchyma of MS brain. (D) The vast majority of CD161 + (green) cells in MS CNS coexpress CD8α (red).

Article Snippet: To characterize T-cell infiltrates in sections of MS brain, the following antibodies against cell surface molecules were used: mouse anti-human CD161 (1:5, 191B8, Miltenyi Biotec, Bergisch Gladbach, Germany), mouse anti-human Vα7.2 (1:5, 3C10, BioLegend, San Diego, CA), mouse anti-human CD8α (1:50, LT8, AbD Serotec, Kidlington, UK; labeled with the Cy3 MAb labeling kit, GE Healthcare, Freiburg, Germany), rabbit anti-human CCR7 (1:800, Y59, Abcam, Cambridge, UK), mouse anti-human CD45RA (1:250, HI100, BioLegend), mouse anti-human CD45RO (1:250, UCHL1, BioLegend), fluorescein isothiocyanate (FITC)-labeled mouse anti-human Vβ1 (1:100, BL37.2, Beckman Coulter, Brea, CA), rabbit anti-human CD3 (1:500, Dako, Glostrup, Denmark), and Alexa Fluor 488–conjugated mouse anti-human CD4 (1:50, RPA-T4, eBioscience, San Diego, CA).

Techniques: Fluorescence, Immunohistochemistry, Staining, Expressing

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: αβ T-cell receptors from multiple sclerosis brain lesions show MAIT cell–related features

doi: 10.1212/NXI.0000000000000107

Figure Lengend Snippet: Analysis of the T-cell receptor (TCR) Vα7.2 repertoire of patient A by pyrosequencing shows oligoclonal T-cell expansions in different samples. Each pie chart represents the total number of nucleotide sequences found in the Vα7.2 repertoire of a certain compartment and each sector represents one distinct T-cell clone. We compared (A) samples from CNS tissue from the biopsy taken in 1996, (B) Vα7.2 + CD161 + mucosal-associated invariant T (MAIT) cells, (C) Vα7.2 + CD161 − cells, (D) CD8 + cells, and (E) CD4 + T cells from peripheral blood taken in 2013 and 2014. For each population we list the designation of the Jα elements and their relative percentage. Clones carrying the MAIT canonical TCR α chain (CAXXDSNYQLIW) are marked in bright blue, and clones containing the noncanonical α chains characterized by Jα12 (CAXXDSSYKLIF) and Jα20 (CAVXXDYKLSF) are marked in dark blue and light blue, respectively. (F) Percentages of identical complementarity determining region 3α amino acid sequences within the TCR Vα7.2 + repertoire between the different samples defined above (A–E). The numbers are percentages indicating how often a particular sequence detected in one sample was also found in another sample. The greatest overlap was between Vα7.2 + CD161 + and CD8 + T cells from peripheral blood, but there was also significant overlap between the CNS sample and the Vα7.2 + CD161 + sample from 2013 to 2014, as highlighted by the red and yellow colors. PBMC = peripheral blood mononuclear cell.

Article Snippet: To characterize T-cell infiltrates in sections of MS brain, the following antibodies against cell surface molecules were used: mouse anti-human CD161 (1:5, 191B8, Miltenyi Biotec, Bergisch Gladbach, Germany), mouse anti-human Vα7.2 (1:5, 3C10, BioLegend, San Diego, CA), mouse anti-human CD8α (1:50, LT8, AbD Serotec, Kidlington, UK; labeled with the Cy3 MAb labeling kit, GE Healthcare, Freiburg, Germany), rabbit anti-human CCR7 (1:800, Y59, Abcam, Cambridge, UK), mouse anti-human CD45RA (1:250, HI100, BioLegend), mouse anti-human CD45RO (1:250, UCHL1, BioLegend), fluorescein isothiocyanate (FITC)-labeled mouse anti-human Vβ1 (1:100, BL37.2, Beckman Coulter, Brea, CA), rabbit anti-human CD3 (1:500, Dako, Glostrup, Denmark), and Alexa Fluor 488–conjugated mouse anti-human CD4 (1:50, RPA-T4, eBioscience, San Diego, CA).

Techniques: Clone Assay, Sequencing