cd73 blocking antibody n 4  (Bio X Cell)

Journal: Blood Cancer Journal

Article Title: Identification and validation of ecto-5' nucleotidase as an immunotherapeutic target in multiple myeloma

doi: 10.1038/s41408-022-00635-3

Figure Lengend Snippet: A Purified MM patient pDCs (from three MM patients) were co-cultured with MM.1S cells (1pDC/5MM) for 48 h, followed by separation of MM.1S cells from pDCs using flow cytometry. Total RNA from MM.1S cells was subjected to RNAseq analysis as in Fig. . Hierarchical clustering analysis of RNAseq data show that pDCs alter purine/pyrimidine nucleotide degradation-related genes including ectoenzyme NT5E/CD73 in MM cells. B CD73 gene enrichment in pDC-MM cell coculture versus MM cells alone [1.34-fold upregulation; n = 3; Adjusted p = 2.04E -05]. C Validation and Quantification of NT5E gene expression by RT-qPCR: MM cells were co-cultured with pDCs for 48 h (in triplicate, n = 3); separated using CD138 antibody by flow sorting, and harvested. Poly RNA was subjected to RT-qPCR using Luna Universal 1-step RT-qPCR kit (New England BioLabs, MA, USA) following manufacturer protocol using an Applied Bioscience 7500 Fast Real-Time PCR System (Thermo Scientific Inc). NT5E/CD73 gene expression was quantified from the raw data using ΔΔCT and utilizing GAPDH as the housekeeping reference gene control under the experimental conditions. The bar graph denotes NT5E. gene expression in MM cells cultured in the presence vs absence of pDCs (mean ± SD; p < 0.05). Inset: Analysis of qPCR amplicons (130 bp) on a 2.5% agarose gel stained with GelRed Nucleic Acid Gel Stain (Biotium Inc, USA). Lane-1: 1 kb DNA ladder; Lane-2 and lane-3: NT5E expression in MM cultured in the absence and presence of pDCs, respectively.

Article Snippet: CD73 blocking antibody (anti-human CD73 Abs) and TLR-7 agonist were obtained from eBiosciences and Invivogen, respectively.

Techniques: Purification, Cell Culture, Flow Cytometry, Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Control, Agarose Gel Electrophoresis, Staining, Expressing

Journal: Blood Cancer Journal

Article Title: Identification and validation of ecto-5' nucleotidase as an immunotherapeutic target in multiple myeloma

doi: 10.1038/s41408-022-00635-3

Figure Lengend Snippet: A pDCs were co-cultured with autologous patient MM cells for 24 h, followed by multicolor flow analyses to assess the pDC-induced change in CD73 expression on MM cells. CD138 + MM cells were examined using flow cytometry, and Median Fluorescence Intensity (MFI) of CD73 expression was determined using anti-CD73 Abs conjugated to AlexaFluor-488, in the presence or absence of pDCs. Left Panel: Representative histograms show CD73 expression in MM cells cultured in the presence (red) and absence (green) of pDCs. [Black histogram: Isotype control Ab]. Right panel: Normalized CD73 expression in MM cells cultured in the presence and absence of pDCs. Data were quantified from the histogram analyses shown in the left panel (mean ± SD; p = 0.008; MFI: 1.2-fold change in pDC-MM vs MM; Data obtained from analysis of seven MM patient BM samples). B pDCs were co-cultured with autologous patient MM cells for 24 h, followed by multicolor flow analyses to assess the changes in CD73 expression on CD303 + MM BM pDCs. Median Fluorescence Intensity (MFI) of CD73 expression was determined using anti-CD73 Abs conjugated to AlexaFluor-647. Representative histograms show CD73 expression in MM BM pDCs cultured in the presence (blue) or absence (green) of MM cells. (Shaded histogram: Isotype control Abs). C MM- BM pDCs were co-cultured with MM cells in the presence and absence of anti-CD73 Abs (0.5 µg/ml) for 2 days; supernatants were collected and analyzed for adenosine levels using a fluorometric adenosine assay kit. The percentage of soluble adenosine in isotype-Ab- versus anti-CD73 Ab-treated MM cells, MM BM pDCs, both alone, or in pDC-MM coculture (mean ± SD, p < 0.05; n = 3) is shown. The quantification of data showing the amount of adenosine in supernatants (pmol/sample) for MM-pDC coculture versus MM cells alone ( p = 0.0167). (Note: The isotype treatment is indicated as a very low level of anti-CD73 Ab in the semi-log plot).

Article Snippet: CD73 blocking antibody (anti-human CD73 Abs) and TLR-7 agonist were obtained from eBiosciences and Invivogen, respectively.

Techniques: Cell Culture, Expressing, Flow Cytometry, Fluorescence, Control

Journal: Blood Cancer Journal

Article Title: Identification and validation of ecto-5' nucleotidase as an immunotherapeutic target in multiple myeloma

doi: 10.1038/s41408-022-00635-3

Figure Lengend Snippet: ( A–D ) MM patient pDCs ( n = 3) were treated with anti-CD73 Ab (0.5 µg/ml) for 24 h, followed by multicolor staining. Expression of pDC activation/maturation markers CD40, CD83, CD86, and HLA-DR were assessed using flow cytometry. Fold change in treated versus untreated pDCs is shown (mean ± SD; p < 0.05; n = 3). E MM patient pDCs ( n = 3) were co-cultured for 3 days with autologous T cells at 1:10 (pDC:T) ratio, in the presence or absence of anti-CD73 Ab (0.5 µg/ml). Viable CD3 + T cells were analyzed for the expression of CD69 activation marker using anti-CD3-FITC and anti-CD69-APC-Cy7 Abs, and quantified by FACS (mean ± SD; P < 0.05, n = 3). [Note: The percentage of CD3 + /CD69 + T cells denotes activated T cells in the presence of anti-CD73 Ab; three myeloma bone marrow (MM BM) samples were used].

Article Snippet: CD73 blocking antibody (anti-human CD73 Abs) and TLR-7 agonist were obtained from eBiosciences and Invivogen, respectively.

Techniques: Staining, Expressing, Activation Assay, Flow Cytometry, Cell Culture, Marker

Journal: Blood Cancer Journal

Article Title: Identification and validation of ecto-5' nucleotidase as an immunotherapeutic target in multiple myeloma

doi: 10.1038/s41408-022-00635-3

Figure Lengend Snippet: A MM patient ( n = 7) BM CD8 + T cells were co-cultured with autologous pDCs (1pDC: 10 T cells), in the presence or absence of anti-CD73 Ab (0.5 µg/ml) for 3 days. After washing to remove anti-CD73 Ab, cells were cultured for 24 h with autologous MM (CD138 + ) cells pre-stained with CellTracker Violet (T/MM; 10:1 ratio), followed by 7-AAD staining, and quantification of CTL-mediated MM cell lysis by FACS. The 7-AAD + populations were gated out and viable 7-AAD negative cells were further gated to quantify 7-AAD negative CellTracker Violet+ cells. Left panel: representative FACS scatter plot showing the decrease in the number of viable 7-AAD negative/Cell Tracker-positive MM cells. Right panel: bar graph shows quantification of CD8 + CTL-mediated MM cell lysis, reflected in decreased CD138 + MM cell viability after anti-CD73 Ab treatment, using data obtained in left panel (mean ± SD; p < 0.05). B MM patient ( n = 3) total BM-MNCs were treated with anti-CD73 Ab (0.5 µg/ml) or isotype-Ab for 2 days, and multicolor flow analysis was utilized to assess MM cell lysis. CD138 + MM cells were quantified by staining with CD138-FITC Ab. Left panel : representative FACS scatter plot showing a decrease in number of viable FITC-positive MM cells after anti-CD73 Ab treatment. Right panel : bar graph shows quantification of CD138 + MM cells in left panel. The fold change was obtained after normalization with control data, and presented as a percentage of viable cells in the presence versus absence of anti-CD73 Ab (mean ± SD; p < 0.05).

Article Snippet: CD73 blocking antibody (anti-human CD73 Abs) and TLR-7 agonist were obtained from eBiosciences and Invivogen, respectively.

Techniques: Cell Culture, Staining, Lysis, Control

Journal: Blood Cancer Journal

Article Title: Identification and validation of ecto-5' nucleotidase as an immunotherapeutic target in multiple myeloma

doi: 10.1038/s41408-022-00635-3

Figure Lengend Snippet: A MM patient ( n = 7) BM C8 + T cells were co-cultured for 3–5 days with autologous pDCs (1 pDC: 10 T cells) in the presence of isotype control Ab, anti-CD73 Ab, TLR-7 agonist, or anti-CD73 Ab plus TLR-7 agonist. Cells were washed and resuspended in a complete medium without drugs, and then cultured with autologous MM cells (10 T: 1 MM cell) pre-stained with CellTrace violet for 24 h, followed by 7-AAD staining and FACS analysis to quantify CTL-mediated MM cell lysis. Left panel: representative FACS scatter plot shows a decrease in number of viable CellTrace Violet-positive MM cells. MM cells were also cultured alone without immune effector cells for 24 h, and viability data obtained from flow analysis was used for normalization and account for spontaneous MM cells death. Right panel: bar graph shows quantification of CD8 + CTL-mediated MM cell lysis, reflected in % Viable MM cells under different treatment conditions, using data obtained in left panel. Percentage of viable MM cells for each treatment versus control (isotype-Ab) is presented. B Bar graph shows normalized NT5E/CD73 and TLR-7 gene enrichment in pDC-MM cell co-cultures versus MM cells alone.

Article Snippet: CD73 blocking antibody (anti-human CD73 Abs) and TLR-7 agonist were obtained from eBiosciences and Invivogen, respectively.

Techniques: Cell Culture, Control, Staining, Lysis

Journal: Blood Cancer Journal

Article Title: Identification and validation of ecto-5' nucleotidase as an immunotherapeutic target in multiple myeloma

doi: 10.1038/s41408-022-00635-3

Figure Lengend Snippet: Schema shows modulation of immunosuppressive adenosine-signaling pathway via CD73 during pDC-T-NK-MM cells interactions, as well as therapeutic utility of targeting CD73 in MM.

Article Snippet: CD73 blocking antibody (anti-human CD73 Abs) and TLR-7 agonist were obtained from eBiosciences and Invivogen, respectively.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Activin-A limits Th17 pathogenicity and autoimmune neuroinflammation via CD39 and CD73 ectonucleotidases and Hif1-α–dependent pathways

doi: 10.1073/pnas.1918196117

Figure Lengend Snippet: CD73 signaling is essential for activin-A–mediated repression of the pathogenic Th17 profile. (A) The expression of Nt5e and Entpd1 was analyzed by qPCR and normalized to Gapdh and Polr2a, in act-A–Th17 or Th17 cells. Each symbol represents the mean ± SEM of duplicate wells and corresponds to one of four independent in vitro experiments. (B) Cumulative data showing CD39 and CD73 expression, gated on CD4+ T cells. Each symbol represents one of four independent in vitro experiments. (C) ATP levels in culture supernatants are shown. Data are mean ± SEM of triplicate wells, each symbol represents one of four independent in vitro experiments. (D) Gene expression was analyzed by qPCR and normalized as above. Each symbol is the mean ± SEM of duplicate wells and corresponds to one of five independent experiments. (E) Cytokine release in culture supernatants. Each symbol is the mean ± SEM of triplicate wells and corresponds to one of five independent in vitro experiments. Statistical significance was obtained by Student’s t test; *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: In another set of experiments, Th17 or act-A–Th17 cells (3 × 10 6 ), generated as above, were treated with anti-CD73 blocking antibody (BioXCell; 20 μg/mL) or IgG control, and then adoptively transferred to C57BL/6- Rag1 −/− mice (day 0) as above.

Techniques: Expressing, In Vitro, Gene Expression

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Activin-A limits Th17 pathogenicity and autoimmune neuroinflammation via CD39 and CD73 ectonucleotidases and Hif1-α–dependent pathways

doi: 10.1073/pnas.1918196117

Figure Lengend Snippet: Blockade of CD73 signaling reverses activin-A–induced suppression of Th17 cell encephalitogenicity. (A) EAE disease scores of C57BL/6-Rag1−/− mice reconstituted with act-A–Th17 or Th17 cells and treated with anti-CD73 antibody or Ig control (experimental protocol in SI Appendix, Fig. S4A). Clinical scores (Left) and regression analysis (Right) are depicted (n = 5 mice per group). Statistical significance was obtained by two-way ANOVA, followed by Bonferroni’s multiple comparisons test. (B) Cytokine levels in spinal cord homogenates. Data are mean ± SEM of triplicate wells; each symbol represents an individual mouse (n = 5 mice per group). Statistical analysis was performed by unpaired Student’s t test; *P < 0.05, **P < 0.01, and ***P < 0.001. Data represent one of two independent experiments.

Article Snippet: In another set of experiments, Th17 or act-A–Th17 cells (3 × 10 6 ), generated as above, were treated with anti-CD73 blocking antibody (BioXCell; 20 μg/mL) or IgG control, and then adoptively transferred to C57BL/6- Rag1 −/− mice (day 0) as above.

Techniques: Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Activin-A limits Th17 pathogenicity and autoimmune neuroinflammation via CD39 and CD73 ectonucleotidases and Hif1-α–dependent pathways

doi: 10.1073/pnas.1918196117

Figure Lengend Snippet: Activin-A–induced STAT3 activation enhances CD73 expression in Th17 cells. (A) Representative FACS plots of Act-A–Th17 or Th17 cells showing pSTAT3 expression. Shaded histogram represents isotype control. Cumulative data are shown as mean ± SEM; each symbol corresponds to one of four independent in vitro experiments. (B) ChIP analyses demonstrating the binding of STAT3 on the Nt5e locus (site 2, +1,700 bp, Left) and on the Entpd1 promoter, at the SRE1 locus (−3,740 bp) (Right). Results are mean ± SEM; each symbol represents the mean ± SEM of duplicate wells and corresponds to one of four independent experiments. (C) Act-A–Th17 cells or Th17 cells were cultured in the presence of STA-21. Gene expression was analyzed by qPCR and normalized to Gapdh and Polr2a. Each symbol represents the mean ± SEM of duplicate wells and corresponds to one of four independent experiments. (D) Cumulative data showing the percentages of CD39+, CD73+, and CD39+CD73+ among CD4+ T cells. Data are mean ± SEM; each symbol corresponds to one of four independent in vitro experiments. (E) IL-10 in culture supernatants. Each symbol represents the mean ± SEM of triplicate wells and corresponds to one of four independent experiments. Statistical analysis was performed by unpaired Student’s t test; *P < 0.05, **P < 0.01 and ***P < 0.001.

Article Snippet: In another set of experiments, Th17 or act-A–Th17 cells (3 × 10 6 ), generated as above, were treated with anti-CD73 blocking antibody (BioXCell; 20 μg/mL) or IgG control, and then adoptively transferred to C57BL/6- Rag1 −/− mice (day 0) as above.

Techniques: Activation Assay, Expressing, Control, In Vitro, Binding Assay, Cell Culture, Gene Expression

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Activin-A limits Th17 pathogenicity and autoimmune neuroinflammation via CD39 and CD73 ectonucleotidases and Hif1-α–dependent pathways

doi: 10.1073/pnas.1918196117

Figure Lengend Snippet: AhR drives activin-A–mediated up-regulation of CD73 and antiinflammatory genes in Th17 cells. (A) Representative immunoblots showing AhR and c-Maf protein levels in act-A–Th17 or Th17 cells. (B) Quantification of relative AhR and c-Maf expression is shown; TATA binding protein (TBP). Data are mean ± SEM; each symbol corresponds to one of four independent in vitro experiments. (C) ChIP analyses demonstrating the binding of AhR and c-Maf on the Nt5e locus. Data are mean ± SEM; each symbol represents one of four independent in vitro experiments. (D) ChIP analyses demonstrating the binding of AhR and c-Maf on the Entpd1 locus and (E) on the Il10 locus. Data are mean ± SEM; each symbol represents one of four independent in vitro experiments. (F) Sequential ChIP analysis demonstrating STAT3 and AhR cobinding on the Il10 conserved noncoding sequence-9 (−9.0 kb) locus. Data are mean ± SEM; each symbol corresponds to one of three independent in vitro experiments. (G) Act-A–Th17 or Th17 cells were differentiated, in the presence of the AhR antagonist, CH-223191 or control (DMSO). Gene expression was analyzed by qPCR and normalized to Gapdh and Polr2a. Each symbol represents the mean ± SEM of duplicate wells and corresponds to one of three independent in vitro experiments. Statistical analysis was performed by unpaired Student’s t test; *P < 0.05, **P < 0.01 and ***P < 0.001.

Article Snippet: In another set of experiments, Th17 or act-A–Th17 cells (3 × 10 6 ), generated as above, were treated with anti-CD73 blocking antibody (BioXCell; 20 μg/mL) or IgG control, and then adoptively transferred to C57BL/6- Rag1 −/− mice (day 0) as above.

Techniques: Western Blot, Expressing, Binding Assay, In Vitro, Sequencing, Control, Gene Expression