Journal: Emerging Microbes & Infections

Article Title: A prophylactic effect of aluminium-based adjuvants against respiratory viruses via priming local innate immunity

doi: 10.1080/22221751.2022.2050951

Figure Lengend Snippet: Type I interferon pathway, rather than type II interferon pathway, is essential for the protective effect of FH-001. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of differentially expressed genes between FH-001-treated lungs and physiological saline-treated lungs from BALB/c mice. The size of the round cycle represents the enriched number of genes; the colour represents the adjusted p -value (p. adjust, Benjamini-Hochberg method). (B) The volcano plot shows the type I interferon pathway associated with differentially expressed genes. The values > 0 represent the genes that were upregulated in FH-001-treated mice. The dashed lines represent the position of ± 1.2-fold. (C) The heatmap shows the expression level of a part of interferon-stimulated genes in lungs from three physiological saline-treated mice (top three rows) and three FH-001-treated mice (bottom three rows). (D) Survival rates of IFNAR-1 blocking antibody (MAR1-5A3, Bio X Cell) treated mice after intranasal administration of 10-fold diluted FH-001 and lethal challenge of FL/2006. The BALB/c mice that were intranasally administered with physiological saline were challenged by FL/2006 and used as a negative control. In addition, an isotype monoclonal antibody (mAb) was also used as a control ( n = 9 mice in FH-001 + MAR1-5A3 treated groups, n = 8 mice for each other groups). (E) Weight change of IFNAR-1 blocking antibody (MAR1-5A3, Bio X Cell) treated mice after intranasal administration of 10-fold diluted FH-001 and lethal challenge of FL/2006 ( n = 9 mice in FH-001 + MAR1-5A3 treated group, n = 8 mice for each other groups). (F) Survival rates of recombinant interferon-α (IFN-α) treated BALB/c mice after a lethal challenge of FL/2006. The BALB/c mice that were intranasally administered with physiological saline were challenged by FL/2006 and used as a negative control ( n = 4 mice per group). (G) Survival rates of IFN-γ neutralizing antibody (XMG1.2, Bio X Cell) treated mice after intranasal administration of 10-fold diluted FH-001 and lethal challenge of FL/2006. The BALB/c mice that were intranasally administered with physiological saline were challenged by FL/2006 and used as a negative control. In addition, a mAb with the same isotype of XMG1.2 was used as isotype control (prepared in-house), n = 6 mice in FH-001 + XMG1.2 treated group, n = 4 mice for each other groups.

Article Snippet: In addition, an isotype monoclonal antibody (mAb) was also used as a control ( n = 9 mice in FH-001 + MAR1-5A3 treated groups, n = 8 mice for each other groups). (E) Weight change of IFNAR-1 blocking antibody (MAR1-5A3, Bio X Cell) treated mice after intranasal administration of 10-fold diluted FH-001 and lethal challenge of FL/2006 ( n = 9 mice in FH-001 + MAR1-5A3 treated group, n = 8 mice for each other groups). (F) Survival rates of recombinant interferon-α (IFN-α) treated BALB/c mice after a lethal challenge of FL/2006.

Techniques: Expressing, Blocking Assay, Negative Control, Recombinant

Journal: Journal for Immunotherapy of Cancer

Article Title: Anti-CD73 antibody activates human B cells, enhances humoral responses and induces redistribution of B cells in patients with cancer

doi: 10.1136/jitc-2022-005802

Figure Lengend Snippet: Mupadolimab directly activates B lymphocytes and induces maturation into antibody secreting plasmablasts in vitro. (A) Purified B cells from 3 to 5 healthy donors were incubated overnight with human IgG 1 isotype control or mupadolimab (10 μg/mL) or anti-IgM microbeads, a positive control for BCR stimulation. Expression of activation markers CD69, CD83, CD86, or MHC-II was measured on viable cells by flow cytometry. Error bars represent mean±SD *p<0.05, **p<0.01, and ****p<0.0001 as determined by t-test. (B) Time dependent increases in the expression of CD27, IgG, IgM, CD38, and CD138 on purified B cells cultured in the presence of mupadolimab or isotype control (1 µg/mL). Mean fluorescence intensity (MFI) was determined for each marker and was normalized to the untreated control for each donor. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 as determined by two-way ANOVA. (C) B cell activation is unique to mupadolimab as other anti-CD73 antibodies do not induce CD69 expression. Expression of CD69 (MFI) was measured by flow cytometry. (D) Representative images of purified B cells cultured with isotype control (left panel) or mupadolimab for 2 days. (E) Purified B cells were incubated overnight with 10 μg/mL human IgG 1 isotype control or mupadolimab or equimolar mupadolimab Fab. CD69 and CD73 were measured on B cells by flow cytometry. (F) Human PBMCs were incubated overnight with a fixed concentration of mupadolimab (10 μg/mL) along with ibrutinib or vehicle control over a range of concentrations. Expression of CD69 on B cells (CD19 POS CD3 NEG ) was measured by flow cytometry. (G) Human PBMCs from healthy donors (n=2) were incubated for 15 min with 10 μg/mL human IgG 1 isotype control, mupadolimab, or anti-IgG+anti IgM, a positive control for BCR stimulation. Phospho-ERK was measured by flow cytometry on B cells, CD4+T cells, and CD8+T cells. (H) Human PBMCs were incubated overnight with 10 μg/mL mupadolimab with or without NECA over a range of concentrations. expression of CD69 on B cells (CD19 POS CD3 NEG ) was measured by flow cytometry and MFI is reported. ANOVA, analysis of variance; BCR, B cell receptor; NECA, 5’-(N-Ethylcarbox-amido) adenosine.

Article Snippet: Human IgG1 isotype control used for in vitro and vivo studies was purchased from Sigma-Aldrich and BioXCell, respectively.

Techniques: In Vitro, Purification, Incubation, Positive Control, Expressing, Activation Assay, Flow Cytometry, Cell Culture, Fluorescence, Marker, Concentration Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Anti-CD73 antibody activates human B cells, enhances humoral responses and induces redistribution of B cells in patients with cancer

doi: 10.1136/jitc-2022-005802

Figure Lengend Snippet: Mupadolimab activates human B cells to secrete immunoglobulin and cytokines associated with B cell activation. (A) Human PBMCs were incubated for 6 days with 1 mg/mL human IgG 1 isotype control or mupadolimab. IgG 1 and IgM secreted into the culture supernatant was quantified by ELISA. IgG-k was not measured as addition of mupadolimab, an IgG1-k antibody, would have confounded the results. Data are represented as mean±SEM of 6 independent experiments, each in duplicate. Purified B cells were incubated for 5 days with 10 µg/mL human IgG 1 isotype control or mupadolimab or anti-IgM microbeads, a positive control for BCR stimulation. Concentration of CCL2 (B), CCL3 (C), CCl4 (D) and CCL22 (E) in supernatant were measured by ELISA. Two representative donors are reported and data are represented as mean±SD of duplicate measurements. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 as determined by two-tailed, unpaired t-test.

Article Snippet: Human IgG1 isotype control used for in vitro and vivo studies was purchased from Sigma-Aldrich and BioXCell, respectively.

Techniques: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Purification, Positive Control, Concentration Assay, Two Tailed Test

Journal: Advanced Science

Article Title: A Single‐Atom Manganese Nanozyme Mn‐N/C Promotes Anti‐Tumor Immune Response via Eliciting Type I Interferon Signaling

doi: 10.1002/advs.202305979

Figure Lengend Snippet: Type I interferon signaling is critical for Mn‐N/C‐mediated anti‐tumor immune response. A) FACS analysis (left) of CFSE and the quantification (right) of OT‐1 T cell proliferation after co‐cultured with DCs sorted from lymph nodes of WT and Ifnar1 −/− MC38‐OVA tumor‐bearing mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 4 per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. B) FACS analysis (below) and the quantification (above) of IFNγ + , Granzyme B + , TNFα + of OT‐1 CD8 + T cells after co‐culture with DCs as A described. Data are shown as mean±SEM ( n = 4 mice per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. C) FACS analysis (above) and the quantification (below) of tumor‐infiltrating CD8 + T cells in WT and Ifnar1 −/− MC38 tumor‐bearing mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 5–6 mice per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. D) FACS analysis (right) and the quantification (left) of IFNγ + , PRF1 + (Perforin), TNFα + of CD8 + T cells in WT and Ifnar1 −/− MC38 tumor‐bearing mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 5–6 mice per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. E) Tumor growth curve of MC38 in WT and Ifnar1 −/− mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 5–6 mice per group). Two‐way ANOVA (mixed model) and Sidak's multiple comparisons test were performed. F) MC38 tumor weight in WT and Ifnar1 −/− mice with vehicle or Mn‐N/C treatment 14 days after tumor cell inoculation. Data are shown as mean±SEM ( n = 5–6 mice per group). G) Kaplan–Meier analysis of WT and Ifnar1 −/− mice survival with vehicle or Mn‐N/C treatment ( n = 5–6 mice per group). * , p < 0.05; ** , p < 0.01; *** , p < 0.001; **** , p < 0.0001; ns, no significance

Article Snippet: Other inhibitors including 1 µ m STING inhibitor H151 (TargetMol, cat#T5674), or 20 µg mL −1 anti‐IFNAR1 neutralizing antibody (BioXCell, cat#BE0241) were added together with Mn‐N/C and H 2 O 2 .

Techniques: Cell Culture, Co-Culture Assay

Journal: Advanced Science

Article Title: A Single‐Atom Manganese Nanozyme Mn‐N/C Promotes Anti‐Tumor Immune Response via Eliciting Type I Interferon Signaling

doi: 10.1002/advs.202305979

Figure Lengend Snippet: A combination of Mn‐N/C and PD‐L1 blockade synergistically suppresses tumor growth. A) Real‐time PCR analysis of PD‐L1 mRNA expression of MC38 tumor cells treated with H 2 O 2 , Mn‐N/C for 24 h. Quantitative data are shown as mean±SEM ( n = 3). One‐way ANOVA and Sidak's multiple comparisons test were performed. B) FACS analysis (left) and the quantification (right) of PD‐L1 levels on the surface of MC38 tumor cells treated with H 2 O 2 , Mn‐N/C, or plus with anti‐IFNAR1 antibody or NAC treatment for 24 h. Quantitative data are shown as mean±SEM ( n = 3). One‐way ANOVA and Sidak's multiple comparisons test were performed. C) Real‐time PCR analysis of PD‐L1 mRNA expression of tumor cells (RFP + ) sorted from MC38‐OVA tumor‐bearing mice with vehicle or Mn‐N/C treatment. Quantitative data are shown as mean±SEM ( n = 3). Two‐tailed unpaired t ‐test was performed for the comparisons between the two groups. D) FACS analysis (left) and the quantification (right) of PD‐L1 levels in CD45 − cells from MC38 tumor‐bearing mice with vehicle or Mn‐N/C treatment. Data are shown as mean±SEM ( n = 6 mice per group). Two‐tailed unpaired t ‐test was performed for the comparisons between the two groups. E) Schematic illustration of a bilateral model of MC38 tumors subcutaneously implanted on C57BL/6 mice and treated with vehicle or Mn‐N/C, or plus with anti‐PD‐L1 antibody. F,G) Tumor growth curve of the first tumor (F) and the second tumor (G) of MC38 in the WT mice treated as E described. Data are shown as mean±SEM ( n = 5 mice per group). Two‐way ANOVA (mixed model) and Sidak's multiple comparisons test were performed. H,I) FACS analysis (left) and the quantification (right) of tumor‐infiltrating CD8 + T cells in the first tumor (H) and the second tumor (I) of MC38 tumor‐bearing mice treated as E described. Data are shown as mean±SEM ( n = 5 mice per group). Two‐tailed unpaired t ‐test was performed for the comparisons between the two groups. J,K) FACS analysis (left) and the quantification (right) of Granzyme B + , PRF1 + , TNFα + of CD8 + T cells in the first tumor (J) and the second tumor (K) of MC38 tumor‐bearing mice treated as E described. Data are shown as mean±SEM ( n = 5 mice per group). Two‐way ANOVA and Sidak's multiple comparisons test were performed. * , p < 0.05; ** , p < 0.01; *** , p < 0.001; **** , p < 0.0001; ns, no significance

Article Snippet: Other inhibitors including 1 µ m STING inhibitor H151 (TargetMol, cat#T5674), or 20 µg mL −1 anti‐IFNAR1 neutralizing antibody (BioXCell, cat#BE0241) were added together with Mn‐N/C and H 2 O 2 .

Techniques: Real-time Polymerase Chain Reaction, Expressing, Two Tailed Test

Journal: Biochemistry

Article Title: Ligand-Guided Selection with Artificially Expanded Genetic Information Systems against TCR-CD3 ε

doi: 10.1021/acs.biochem.9b00919

Figure Lengend Snippet: Workflow of AEGIS–LIVE–LIGSa aAEGIS–LIVE is first employed against Jurkat.E6 cells until a partial enrichment of the AEGIS DNA aptamer library is achieved. Next, the partially enriched AEGIS–LIVE pool is divided into fractions. The first fraction, which is partially enriched with respect to Jurkat.e6 cells, is amplified via PCR and subjected to Illumina-HT sequencing. An excess of mAb (Isotype, OKT3, UCHT1, or CD 28) is then introduced into each remaining fraction, which was pre-incubated with target cells to selectively elute potential aptamers that would tend to bind to the mAb’s cognate epitope. The sequences eluted by each mAb are amplified via PCR, converted to the four-letter alphabet, and subjected to Illumina-HT sequencing. Finally, sequences obtained from sequencing of each fraction were analyzed using FASTAptamer and Galaxy program, and on the basis of set criteria, specific aptamer candidates against respective epitopes on the target cells are identified.

Article Snippet: Anti-CD3 monoclonal antibodies UCHT1 (mouse anti-human, isotype IgG1, catalog no. BE0231), OKT3 (mouse anti-human, isotype IgG2a, catalog no. BE0001-2)- and the anti-CD28 monoclonal antibody (mouse anti-human, isotype IgG2a, clone 9.3, catalog no. BE0248) were obtained from BioXCell.

Techniques: Amplification, Sequencing, Incubation

Journal: Biochemistry

Article Title: Ligand-Guided Selection with Artificially Expanded Genetic Information Systems against TCR-CD3 ε

doi: 10.1021/acs.biochem.9b00919

Figure Lengend Snippet: Post-LIGS analysis of tenth-round AEGIS–LIVE pool binding. (A) Analysis of tenth-round AEGIS–LIVE pool affinity with Jurkat.E6 cells. (B) Evaluation of tenth-round AEGIS–LIVE pool binding with Jurkat cells in the presence of an isotype control, anti-CD28, OKT3, and UCHT1. The normalized binding on the y-axis was obtained by first calculating specific binding values compared to the unselected control library as (fluorescence signal for round 10−fluorescence signal for controlfluorescence for control)×100. Specific binding was then normalized against the total binding obtained in the presence of the isotype control antibody. Then the binding values (median fluorescence intensity of respective histograms) corresponding to each antibody were normalized to total binding. (C) Post-LIGS analysis of tenth-round AEGIS–LIVE pool binding to Jurkat cells in the presence of the isotype control antibody (blue), the anti-CD28 antibody (green), the UCHT1 antibody (orange), or the OKT3 antibody (red).

Article Snippet: Anti-CD3 monoclonal antibodies UCHT1 (mouse anti-human, isotype IgG1, catalog no. BE0231), OKT3 (mouse anti-human, isotype IgG2a, catalog no. BE0001-2)- and the anti-CD28 monoclonal antibody (mouse anti-human, isotype IgG2a, clone 9.3, catalog no. BE0248) were obtained from BioXCell.

Techniques: Binding Assay, Control, Fluorescence

Journal: Biochemistry

Article Title: Ligand-Guided Selection with Artificially Expanded Genetic Information Systems against TCR-CD3 ε

doi: 10.1021/acs.biochem.9b00919

Figure Lengend Snippet: Bioinformatics analysis of LIVE pools and LIGS pools to identify AEGIS DNA ligands. (A) Enrichment, as defined by (1−number of unique sequencestotal number of sequences)×100, was calculated for AEGIS–LIVE pools from the fifth, eighth, and tenth rounds, and the enrichment was calculated for LIGS pools that originated from isotype, OKT3, UCHT1, and anti-CD20 mAbs. The fold enrichment ratios against isotype control antibody for the (B) OKT3, (C) UCHT1, and (D) anti-CD28 mAbs were plotted as a function of the normalized read counts (RPM) for individual sequences. Sequences showing a fold enrichment value of >2 (above the solid line) were considered as specific against each mAb. (E) Flowchart summarizing the GALAXY workflow sequence files for OKT3 and UCHT1 to identify specific hit AEGIS DNA ligand candidates.

Article Snippet: Anti-CD3 monoclonal antibodies UCHT1 (mouse anti-human, isotype IgG1, catalog no. BE0231), OKT3 (mouse anti-human, isotype IgG2a, catalog no. BE0001-2)- and the anti-CD28 monoclonal antibody (mouse anti-human, isotype IgG2a, clone 9.3, catalog no. BE0248) were obtained from BioXCell.

Techniques: Control, Sequencing

Journal: Biochemistry

Article Title: Ligand-Guided Selection with Artificially Expanded Genetic Information Systems against TCR-CD3 ε

doi: 10.1021/acs.biochem.9b00919

Figure Lengend Snippet: Specificity analyses of the JZPO-10 aptamer. (A) Flow cytometric analyses for binding of the JZPO-10 aptamer against Jurkat.E6 (left), MOLT-3 (middle), and Toledo (right) cells. (B) Overall conclusion from three independent binding assays against each cell line. The percentage of specific binding on the y-axis for each cell line was determined as (aptamer−randomrandom)×100. Statistical analysis was performed in GraphPad Prism using one-way ANOVA and Tukey’s multiple-comparison test (****p < 0.0001). (C) Binding analysis of the JZPO-10 aptamer against wild-type Jurkat cells used for CRISPR-Cas9 (left) and against a CD3ε/TCRα double-knockout Jurkat cell line generated by CRISPR-Cas9 (right) (Figure S3 shows antibody staining). (D) Overall conclusion from four independent binding analyses using wild-type and knockout Jurkat cells. The percentage of specific binding on the y-axis was calculated using the formula mentioned above. Statistical significance was evaluated by an unpaired t test using GraphPad Prism (****p < 0.0001).

Article Snippet: Anti-CD3 monoclonal antibodies UCHT1 (mouse anti-human, isotype IgG1, catalog no. BE0231), OKT3 (mouse anti-human, isotype IgG2a, catalog no. BE0001-2)- and the anti-CD28 monoclonal antibody (mouse anti-human, isotype IgG2a, clone 9.3, catalog no. BE0248) were obtained from BioXCell.

Techniques: Binding Assay, Comparison, CRISPR, Double Knockout, Generated, Staining, Knock-Out

Journal: Biochemistry

Article Title: Ligand-Guided Selection with Artificially Expanded Genetic Information Systems against TCR-CD3 ε

doi: 10.1021/acs.biochem.9b00919

Figure Lengend Snippet: Analyses of cellular and epitope specificity of two truncated variants of the JZPO-10 aptamer. (A) Binding analysis of JZPO-10.3 and JZPO-10.4 aptamers against wild-type Jurkat (top), CD3ε/TCRα double-knockout Jurkat (middle), and MOLT-3 (bottom) cells. (B) Flow cytometric analyses of competitive binding experiments of the JZPO-10.4 aptamer in the presence and absence of OKT3. The JZPO10.4 aptamer was incubated with Jurkat E.6 cells in the presence of either the control antibody against CD28 (blue) or OKT3 mAb (red; Figure S5 shows the corresponding secondary antibody staining). (C) Overall conclusion from three independent mAb competitive binding experiments for the JZPO-10.4 aptamer. Statistical significance was evaluated using an unpaired t test in GraphPad Prism (***0.0001 ≤ p ≤ 0.001).

Article Snippet: Anti-CD3 monoclonal antibodies UCHT1 (mouse anti-human, isotype IgG1, catalog no. BE0231), OKT3 (mouse anti-human, isotype IgG2a, catalog no. BE0001-2)- and the anti-CD28 monoclonal antibody (mouse anti-human, isotype IgG2a, clone 9.3, catalog no. BE0248) were obtained from BioXCell.

Techniques: Binding Assay, Double Knockout, Incubation, Control, Staining