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: 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

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: 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: PLoS Pathogens

Article Title: FCRL5 Delineates Functionally Impaired Memory B Cells Associated with Plasmodium falciparum Exposure

doi: 10.1371/journal.ppat.1004894

Figure Lengend Snippet: ( A ) Sorted transitional cells (CD19 + CD10 + ), CD20 + atMBCs (IgG + CD21 - CD27 - CD19 + ), classical MBCs (IgG + CD21 + CD27 + CD19 + ), and CD27 - plasmablasts (CD20 - IgG + CD21 - CD27 - CD19 + ) were cultured on anti-IgG ELISpot plates for 18 h without additional stimulation. ( B ) Gating strategy and frequencies of CD38 hi cells in the above plasmablast gating strategy.

Article Snippet: Isotype controls included mouse IgG1 (clone MOPC-21) (Tonbo Biosciences), and IgG2a (clone MOPC-173) and IgG2b (clone MPC-11) (BioLegend).

Techniques: Cell Culture, Enzyme-linked Immunospot

Journal: PLoS Pathogens

Article Title: FCRL5 Delineates Functionally Impaired Memory B Cells Associated with Plasmodium falciparum Exposure

doi: 10.1371/journal.ppat.1004894

Figure Lengend Snippet: ( A ) Surface expression, expressed as median fluorescence intensity (MFI), of CD85d, CD120b, CD360, CD11c, and IgG (BCR) on IgG + atMBCs and IgG + classical MBCs. Lines between symbols denote MBC subsets from the same subject. Wedges represent means. ( B ) Labeling of SVT2 mouse fibroblast cell lines that express full-length human FCRL4 or FCRL5 protein by monoclonal antibodies 2A6, 1A3, and 7D11. ( C ) Labeling of human atMBCs with monoclonal antibodies 2A6, 1A3, and 7D11. ( D ) Isotype-subtracted MFI of FCRL family member expression (“Net MFI”) on atypical and classical MBCs from highly P . falciparum -exposed individuals. Statistical significance was determined using the Wilcoxon signed-rank test. *, p < 0.05; **, p < 0.01

Article Snippet: Isotype controls included mouse IgG1 (clone MOPC-21) (Tonbo Biosciences), and IgG2a (clone MOPC-173) and IgG2b (clone MPC-11) (BioLegend).

Techniques: Expressing, Fluorescence, Labeling, Bioprocessing

Journal: PLoS Pathogens

Article Title: FCRL5 Delineates Functionally Impaired Memory B Cells Associated with Plasmodium falciparum Exposure

doi: 10.1371/journal.ppat.1004894

Figure Lengend Snippet: ( A ) Representative plot showing heterogeneous expression of FCRL5 on IgG + atypical MBCs. Individual FCRL5 + atypical and classical MBC frequencies were determined using gates set with a “fluorescence minus one” control with IgG2b isotype control antibody. ( B ) Proportion of atypical and classical IgG + MBCs expressing FCRL5. Reported frequencies have been subtracted for isotype-labeled background. ( C ) Median fluorescence intensity (MFI) of surface markers on FCRL5 + vs. FCRL5 - atMBCs and FCRL5 + vs. FCRL5 - classical MBCs. Statistical significance was determined using the Wilcoxon signed-rank test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Isotype controls included mouse IgG1 (clone MOPC-21) (Tonbo Biosciences), and IgG2a (clone MOPC-173) and IgG2b (clone MPC-11) (BioLegend).

Techniques: Expressing, Fluorescence, Control, Labeling

Journal: PLoS Pathogens

Article Title: FCRL5 Delineates Functionally Impaired Memory B Cells Associated with Plasmodium falciparum Exposure

doi: 10.1371/journal.ppat.1004894

Figure Lengend Snippet: Sorted FCRL5 + and FCRL5 - , atypical (CD20 + CD21 - CD27 - IgG + ) and classical (CD20 + CD21 + CD27 + IgG + ) MBCs were stimulated for 4 days with CpG, F(ab’) 2 anti-IgG, and autologous T cells. IgG-secreting cells were detected by IgG ELISpot and are reported as the number of IgG secreting cells per 1000 cells sorted on day 0. ASC, antibody-secreting cells. Statistical significance was determined using the Wilcoxon signed-rank test. *, p < 0.05.

Article Snippet: Isotype controls included mouse IgG1 (clone MOPC-21) (Tonbo Biosciences), and IgG2a (clone MOPC-173) and IgG2b (clone MPC-11) (BioLegend).

Techniques: Enzyme-linked Immunospot