Journal: Diabetologia

Article Title: Phenotypically distinct anti-insulin B cells repopulate pancreatic islets after anti-CD20 treatment in NOD mice

doi: 10.1007/s00125-019-04974-y

Figure Lengend Snippet: Anti-insulin B cells repopulate pancreatic islets more rapidly than insulin-negative B cells after anti-CD20 treatment. Groups of VH125.hCD20/NOD mice, aged 6–8 weeks old, were injected with 2H7 anti-CD20 or IgG isotype control. Spleen, PLNs and pancreatic islets were analysed for insulin-positive and insulin-negative B cells at 8 weeks and 12 weeks post depletion by flow cytometry. ( a – f ) No. of cells from IgG control-treated (black circles) and 2H7-treated (grey squares) mice for insulin-negative B cells ( a – c ) and insulin-positive B cells ( d – f ) from spleen ( a , d ) PLNs ( b , e ) and islets ( c , f ). ( g – i ) Percentage of B cells repopulated at 8 and 12 weeks after treatment from spleen ( g ), PLNs ( h ) and islets ( i ) of mice shown in ( a – f ). Percentages were calculated as individual numbers from each 2H7-treated mouse / mean number from all IgG control antibody-treated mice. Horizontal lines represent the median value. Data represent three independent experiments. At 8 weeks, n = 7 (spleen), n = 7 (PLNs) and n = 9 (islets) for control IgG-treated mice and n = 10 (spleen), n = 9 (PLNs) and n = 12 (islets) for 2H7-treated mice. At 12 weeks, n = 8 (spleen), n = 6 (PLNs) and n = 7 (islets) for control IgG and n = 11 (spleen), n = 7 (PLNs) and n = 11 (islets) for 2H7-treated mice. * p < 0.05 (one-way ANOVA)

Article Snippet: Female VH125.hCD20/NOD mice, 6–8 weeks of age were chosen at random to receive either anti-hCD20 antibody (clone 2H7; Bio-XCell, West Lebanon, NH, USA) or control IgG2b antibody (clone MPC-11; Bio-XCell [ , , ]), as described previously [ ].

Techniques: Injection, Control, Flow Cytometry

Journal: Diabetologia

Article Title: Phenotypically distinct anti-insulin B cells repopulate pancreatic islets after anti-CD20 treatment in NOD mice

doi: 10.1007/s00125-019-04974-y

Figure Lengend Snippet: CD138 int anti-insulin B cells are enriched in pancreatic islets after anti-CD20 treatment. Groups of 6- to 8-week-old VH125.hCD20/NOD mice were injected with 2H7 anti-CD20 or IgG isotype control. Groups of mice ( n = 2 or 3 per group) were pooled and insulin + B cells from pancreatic islets were analysed for four different populations based on CD138 expression: CD138 − (blue); CD138 int IgM + (orange); CD138 int IgM lo (grey) and CD138 hi IgM lo (red). ( a , b ) Representative flow plots showing gating on live CD3 − CD11b − CD11c − ( a ) and graph showing the overall percentages of the four different populations ( b ). ( c , d ) Representative flow plots showing insulin − CD19 + , insulin + CD19 + and insulin + CD19 − cells ( c ) and graph showing the overall percentages of these cells ( d ); 2H7 (black circles), IgG (grey circles). ( e ) Representative flow plots showing CD138 and IgM expression in insulin + CD19 + and insulin + CD19 − cells. ( f , g ) Graphs showing CD138 and IgM populations on insulin + CD19 + ( f ) and insulin + CD19 − cells ( g ) ( n = 5 groups for control IgG treatment; n = 4 groups for 2H7 treatment). Horizontal lines represent the median values. Data represent two independent experiments. * p < 0.05 (one-way ANOVA)

Article Snippet: Female VH125.hCD20/NOD mice, 6–8 weeks of age were chosen at random to receive either anti-hCD20 antibody (clone 2H7; Bio-XCell, West Lebanon, NH, USA) or control IgG2b antibody (clone MPC-11; Bio-XCell [ , , ]), as described previously [ ].

Techniques: Injection, Control, Expressing

Journal: Cell Research

Article Title: Targeting ATAD3A-PINK1-mitophagy axis overcomes chemoimmunotherapy resistance by redirecting PD-L1 to mitochondria

doi: 10.1038/s41422-022-00766-z

Figure Lengend Snippet: a Left, immunostaining of PD-L1 (green) and TOM20-labeled mitochondria (red) in BT549 human TNBC cells with or without ATAD3A-knockdown (shATAD3A#1 and shATAD3A#2). Scale bars, 20 μm and 2 μm (inset). Right, the percentage of PD-L1 co-localized with TOM20 ( n = 5 fields, t -test). b Immunoblot of PD-L1 in the cytoplasm and mitochondria of control and ATAD3A-knockdown BT549 cells. TOM20 and Tubulin were used as mitochondria and cytoplasm protein controls, respectively. Cyto, cytoplasm; mito, mitochondria. c Flow cytometry (left) and quantification (right) of surface PD-L1 in control and ATAD3A-knockdown BT549 cells ( n = 3, one-way ANOVA). d Venn diagram depicting overlapped genes for the interaction protein of ATAD3A set (BioGRID, RP5-832C2.1), the protein localization to mitochondrion set (GOBP 0070585) and the intrinsic component of mitochondrial membrane (GOCC 0098573). e Immunoblot of PINK1 in control and ATAD3A-knockdown BT549 cells. f Immunoblot of PD-L1 and PINK1 in HEK293T cells overexpressing PD-L1 (OE-PD-L1) and control cells (OE-Control), assessed after immunoprecipitation with immunoglobulin G (IgG) or antibody to PINK1. g Protein direct interaction analysis of the intracellular domain of PD-L1 (ICD) and PINK1 in vitro. Purified Flag-labeled full-length PINK1 was incubated with Biotin-labeled PD-L1 ICD domain, followed by streptavidin pull-down and immunoblot. h Schematic diagram of Flag-labeled full-length (FL) and truncated mutants with indicated domains (amino acids 1–155, amino acids 156–320, amino acids 321–509, amino acids 510–581) of PINK1. MTS, mitochondrial targeting sequence; N-lobe, kinase domain N; C-lobe, kinase domain C; CTD, C-terminal domain. i Protein direct interaction analysis of the intracellular domain of PD-L1 (ICD) and truncated PINK1 mutants in vitro. Purified Flag-labeled full-length and truncated PINK1 were incubated with Biotin-labeled PD-L1 ICD domain, followed by streptavidin pull-down and immunoblot. The estimated size of PINK1-4 (amino acids 510–581) which did not express in HEK293T cells was labeled with asterisk. j Immunoblot of PD-L1 in the cytoplasm and mitochondria of MDA-MB-231 cells with or without PINK1-knockdown (shPINK1#1 and shPINK1#2). TOM20 and Tubulin were used as mitochondria and cytoplasm protein controls. Cyto, cytoplasm; mito, mitochondria. k Left, co-localization of PD-L1 (green) and TOM20 (red) in control, ATAD3A knockdown, PINK1 knockdown or ATAD3A and PINK1 double knockdown BT549 cells. Scale bars, 20 μm and 2 μm (inset). Right, the percentage of PD-L1 co-localized with TOM20 ( n = 5 fields, one-way ANOVA). l Immunoblot of PD-L1 in the cytoplasm and mitochondria of control, ATAD3A-knockdown, PINK1-knockdown or ATAD3A and PINK1 double knockdown BT549 cells. m Immunoblot of indicated proteins in PD-L1-transfected HEK293T cells with or without PINK1 overexpression. n Immunoblot of PD-L1 in control and PINK1-knockdown (shPINK1#1 and shPINK1#2) BT549 cells. o Immunoblot of PD-L1 in BT549 cells transfected with control shRNA or shATAD3A (shATAD3A#1 and shATAD3A#2). p Immunoblot of total PD-L1 in control, ATAD3A-knockdown, PINK1-knockdown or ATAD3A and PINK1 double knockdown BT549 cells. q Immunoblot of PD-L1 in control and ATAD3A-knockdown BT549 cells treated with 20 μM CHX for indicated times. h, hours. r Quantification of PD-L1 intensity in immunoblot in control and ATAD3A-knockdown BT549 cells. s Immunoblot of PD-L1 in control and ATAD3A-knockdown MDA-MB-231 cells incubated with 20 nM BafA1 for indicated times. h, hours. Data are representative of at least two independent experiments and are shown as means ± SD. See also Supplementary information, Figs. and .

Article Snippet: Mouse IgG2b (APC, clone MPC-11, BioLegend) and rat IgG2b (APC, clone RTK4530, BioLegend) were used as controls respectively.

Techniques: Immunostaining, Labeling, Knockdown, Western Blot, Control, Flow Cytometry, Membrane, Immunoprecipitation, In Vitro, Purification, Incubation, Sequencing, Transfection, Over Expression, shRNA

Journal: Cell Research

Article Title: Targeting ATAD3A-PINK1-mitophagy axis overcomes chemoimmunotherapy resistance by redirecting PD-L1 to mitochondria

doi: 10.1038/s41422-022-00766-z

Figure Lengend Snippet: a – h BALB/c mice were inoculated orthotopically with 5 × 10 4 4T1 cells transfected with control shRNA (shControl) or shRNA for Atad3a (shAtad3a#1 and shAtad3a#2). a , b The endpoint tumor images ( a ) and volume ( b ) of tumors formed by control and Atad3a-knockdown cells in BALB/c mice ( n = 6, one-way ANOVA). c Left, IHC staining of Atad3a and PD-L1 on serial sections of tumors formed by control and Atad3a-knockdown cells. Scale bars, 50 μm. Right, IHC score of Atad3a in control and Atad3a-knockdown tumors ( n = 6 fields, t -test). d IHC score of PD-L1 in control and Atad3a-knockdown tumors ( n = 6 fields, t -test). e Quantification of the percentage of tumor-infiltrating CD8 + T cells in tumors formed by control and Atad3a-knockdown cells by flow cytometry ( n = 5, t -test). f Quantification of the percentages of tumor-infiltrating IFNγ + CD8 + T cells and IFNγ + CD4 + T cells by flow cytometry ( n = 5, t -test). g Ratio of CD8 + cytotoxic T lymphocytes to CD4 + CD25 + Foxp3 + T reg cells ( n = 5, t -test). h Quantification of the percentages of PD-1 + TIM-3 + CD8 + T cells (left) and PD-1 + TIM-3 + CD4 + T cells (right) by flow cytometry ( n = 5, t -test). i – m BALB/c mice were inoculated orthotopically with 5 × 10 4 4T1 cells transfected with control shRNA (shControl) or shRNA specific for Atad3a (shAtad3a), Pink1 (shPink1) or both (shAtad3a + shPink1). i , Left, the endpoint images of tumors formed by control, Atad3a-knockdown, Pink1-knockdown or Atad3a and Pink1 double knockdown 4T1 cells in BALB/c mice. Right, immunoblot of Atad3a and Pink1 in these 4T1 cells. j The volume of tumors mentioned above ( n = 6, one-way ANOVA). k Quantification of the percentage of tumor-infiltrating CD8 + T cells by flow cytometry ( n = 5, one-way ANOVA). l Quantification of the percentage of tumor-infiltrating IFNγ + CD8 + T cells by flow cytometry ( n = 5, one-way ANOVA). m Quantification of the percentage of PD-1 + TIM-3 + CD8 + T cells by flow cytometry ( n = 5, one-way ANOVA). n – s 4T1 tumors formed by control and Atad3a-knockdown cells were established orthotopically in BALB/c mice and received vehicle, anti-PD-L1 antibody (PD-L1 mAb), paclitaxel (PTX) or combined anti-PD-L1 antibody with paclitaxel treatment (PD-L1 mAb + PTX). IgG2b and saline were used as controls. n Experimental protocol. o , p The endpoint tumor images ( o ) and the volume ( p ) of tumors ( n = 6, one-way ANOVA). q – s Quantification of the percentages of tumor-infiltrating CD8 + T cells ( q ), IFNγ + CD8 + T cells ( r ) and PD-1 + TIM-3 + CD8 + T cells ( s ) in 4T1 tumors formed by control and Atad3a-knockdown cells received treatments as described above, determined by flow cytometry ( n = 5, one-way ANOVA). t Schematic model. Patients with PD-L1-positive TNBC could be divided into two groups based on ATAD3A expression. Patients with ATAD3A-high tumors might respond more poorly to ICIs plus paclitaxel therapy, and inhibition of ATAD3A is required to improve clinical outcome. Patients with ATAD3A-low tumors might benefit significantly from ICIs plus paclitaxel combination therapy. See also Supplementary information, Figs. – .

Article Snippet: Mouse IgG2b (APC, clone MPC-11, BioLegend) and rat IgG2b (APC, clone RTK4530, BioLegend) were used as controls respectively.

Techniques: Transfection, Control, shRNA, Knockdown, Immunohistochemistry, Flow Cytometry, Western Blot, Saline, Expressing, Inhibition

Journal: Cancer immunology, immunotherapy : CII

Article Title: Identification and characterization of an alternative cancer-derived PD-L1 splice variant

doi: 10.1007/s00262-018-2284-z

Figure Lengend Snippet: a) PD-1 binding capacity of IgG1-FC (negative control), rhPD-L1-FC (positive control) or sec-PD-L1-long-FC was measured using a functional ELISA, where mean OD was measured at varying concentrations of IgG1-FC, rhPD-L1-FC or sec-PD-L1-long-FC protein. Kd values are displayed. The corresponding Scatchard graph displaying the differences in slope (−1/Kd) of rhPD-L1-FC (medium gray) or sec-PD-L1-long-FC (light gray) is shown as an inset. Data is from two separate experiments. b) The ability of PD-L1 and PD-1 neutralizing antibodies (10ug/ml) to block PD-1 binding to rhPD-L1-FC and sec-PD-L1-long-FC was tested with a functional ELISA. An IgG1 isotype control antibody was used as a negative control. Normalized mean OD values compared to incubation with the appropriate IgG1 isotype control conditions are plotted. Data is from two separate experiments. A two-way ANOVA was used. The mean and SEM are plotted. c, d) Primary human T cell blasts were incubated with IgG1-FC (negative control), rhPD-L1-FC (positive control) or sec-PD-L1-long-FC at either 20ug/ml or 40ug/ml in the presence of anti-CD3 for 24 hours, and media was harvested to quantify c) IL-2 and d) IFNg with ELISA. Samples were run in duplicate. N=5. Normalized protein levels compared to the appropriate IgG1-Fc control are plotted. A one-way ANOVA was used. The mean and SEM are plotted. *p<0.05, **p<0.01, ***p<0.001

Article Snippet: For PD-L1 surface staining of cancer cell lines, cells were stained as described with the following antibodies: anti-human CD274-APC (1:100, clone 29E.2A3, mouse IgG2b, κ, Biolegend, #329707) or isotype control (1:100, clone MPC-11, mouse mouse IgG2b, κ, Biolegend, #400319.

Techniques: Binding Assay, Negative Control, Positive Control, Functional Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay, Incubation

Journal: Cell

Article Title: B cells and T follicular helper cells mediate response to checkpoint inhibitors in high mutation burden mouse models of breast cancer.

doi: 10.1016/j.cell.2019.10.028

Figure Lengend Snippet: (A) RNA-seq signatures for sensitive tumors at 7 days (5mm= day 0/ treatment initiation) without or with anti-PD1/anti-CTLA4 therapy. (B) Flow cytometry results for CD8+ cells and CD4+ using memory markers (Cd44, Cd62L). (C) Flow cytometry of tumor infiltrating B cells with or without ICI therapy. On the right shows staining for B cells gated for activation markers. (D) Quantification of flow cytometry for activated B cells (B220+, Cd19+ or Cd20+, MHC II +, Cd80+ or Cd86+). (E) IHC staining for IgG-kappa chain in KPB25Luv tumors. (F) IgG binding assay showing serum-IgG binding (Fitc+) to KPB25Luv cells. (G) Quantification of Fitc+ cells in IgG binding assay for KP25Luv cells and off-target binding. (H) Quantification of Fitc+ IgG binding assay for T11-Apobec cells following reabsorption on off-target cells. In boxplots, bars signify the mean and standard deviation. The p-values are two-tailed from unmatched T-tests. All tumors collected after 7days of treatment or non-treatment.

Article Snippet: Mouse IgG2b Isotype Control (clone MPC-11) , Bioxcel , Cat: BE0086; RRID: AB_1107791.

Techniques: RNA Sequencing, Flow Cytometry, Staining, Activation Assay, Immunohistochemistry, Binding Assay, Standard Deviation, Two Tailed Test

Journal: Cell

Article Title: B cells and T follicular helper cells mediate response to checkpoint inhibitors in high mutation burden mouse models of breast cancer.

doi: 10.1016/j.cell.2019.10.028

Figure Lengend Snippet: (A) Flow cytometry for B cells in KPB25Luv tumors after 7 days of ICI and CD4+ T cell depletion. (B) Quantification of results from A. (C) X-Y plot of IgG and CIBERSORT Tfh T cell signatures in mRNA-seq of sensitive tumors at day 7. (D) Boxplot of CIBERSORT Tfh T cell signature levels in sensitive tumors (mRNA-seq) at day 7. (E) X-Y plot of IgG signature and Il21 mRNA in mRNA-seq of sensitive tumors at day 7. (F) Boxplot of Il21 mRNA levels in sensitive tumors (mRNA-seq) at day 7. (G) Flow cytometry results for activated B cells in T11-Apobec & KPB25Luv tumors during Tfh/IL21 blockade. (H) IHC staining for IgG-kappa chain in KPB25Luv tumors during Tfh/IL21 blockade. (I) Survival for T11-Apobec bearing mice during ICI therapy and Tfh/IL21 blockade. (J) Survival for KPB25Luv bearing mice during ICI therapy and Tfh/IL21 blockade. (K) Western blot for serum IgG in Igmi and Balbc mice with T11-Apobec tumors. The blue bars mark Igmi mouse sera, purple note Balbc sera. (L) 21 day acute response in Igmi and Balbc control mice with T11-Apobec tumors. (M) Survival of Igmi mice withT11-Apobec tumors and treated with anti-PD1/anti-CTLA4 therapy in contrast to Balbc controls. (N) Survival in KPB25Luv tumor bearing mice treated with ICI therapy or ICI therapy with CD16/32 blockade . In Kaplan-Meier plots, p-values are from Log-rank (Mantel-Cox) tests. Boxplots show the mean and standard deviation. The p-values are two-tailed from standard T-tests. In X-Y plots, p-values were determined by linear regression analysis. The asterisks denote significance (***, p<0.0001, *, P<0.05).

Article Snippet: Mouse IgG2b Isotype Control (clone MPC-11) , Bioxcel , Cat: BE0086; RRID: AB_1107791.

Techniques: Flow Cytometry, Immunohistochemistry, Western Blot, Control, Standard Deviation, Two Tailed Test

Journal: Cell

Article Title: B cells and T follicular helper cells mediate response to checkpoint inhibitors in high mutation burden mouse models of breast cancer.

doi: 10.1016/j.cell.2019.10.028

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse IgG2b Isotype Control (clone MPC-11) , Bioxcel , Cat: BE0086; RRID: AB_1107791.

Techniques: Control, Virus, Recombinant, Staining, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Microarray, Plasmid Preparation, Software, Membrane, Transfection

Journal: Cell

Article Title: B cells and T follicular helper cells mediate response to checkpoint inhibitors in high mutation burden mouse models of breast cancer.

doi: 10.1016/j.cell.2019.10.028

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse IgG2b Isotype Control (clone MPC-11) , Bioxcel , Cat: BE0086; RRID: AB_1107791.

Techniques: Control, Virus, Recombinant, Staining, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Microarray, Plasmid Preparation, Software, Membrane, Transfection

Journal: Cytometry

Article Title: A human receptor occupancy assay to measure anti‐PD ‐1 binding in patients with prior anti‐PD ‐1

doi: 10.1002/cyto.a.24334

Figure Lengend Snippet: Validation of RG7769 receptor occupancy (RO) assay for whole‐blood T cell RO analysis. (A) Gating strategy for RO calculation. I. In all conditions, single lymphocytes were identified by excluding cell doublets via FSC‐H versus FSC‐W followed by SSC‐H versus SSC‐W. II. Total lymphocytes were gated on via CD45 staining versus SSC‐A. III. Within the CD45 + compartment, T cells were identified via CD3 expression. A non‐T cell gate (NOT‐CD3 + ) was set. IV. Within total CD3 + T cells, CD4 + (Q3) and CD8 + single‐positive (Q1) T cells were identified. V. In the non‐T cell population, NK cells were identified via CD56 versus CD16 (QI‐III). Total NK cells were defined as CD56 + CD16 +/− . Lower right: Comparing the anti‐PGLALA‐PE staining intensity of CD3 + T cells, CD4 + and CD8 + T cells pulsed with RG7769. Orange: 1 μg/ml, red: Saturation (100 μg/ml); gray: Negative control (1000 ng/ml + mIgG 2b ‐isotype‐PE). (B) Dose–response of RG7769 binding to healthy donor lymphocytes. Using the gating strategy in A, CD45 + lymphocyte single cells from blood pulsed ex vivo with RG7769 (0 ng/ml➔100 ng/ml), were identified. The control group (0 ng/ml) was stained with mIgG 2b ‐PE isotype, cells with RG7769 with anti‐PGLALA‐PE. Five thousand concatenated CD45 + events were clustered according to CD3, CD4, CD8, CD56 and CD16 by UMAP. Left: Cell lineages identified using FlowSOM include CD4 + T cells, CD8 + T cells, CD4 − CD8 − unconventional T cells as well as NK cell subsets. Right: PE + (RG7769 + ) T cells (blue) [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: After washing with PBS, tube 1 (negative control) was stained with CD3 FITC (SK7, BioLegend), mIgG 2b PE (MPC‐11), BD Biosciences, CD8‐APC (SK1, BioLegend), CD45‐APC‐H7 (2D1, BD Bioscience), CD56‐BV421 (HCD56, BioLegend), CD4‐BV510 (SK3, BioLegend), CD16‐BV605 (3G8), BD Bioscience).

Techniques: Staining, Expressing, Negative Control, Binding Assay, Ex Vivo