Journal: PLOS Pathogens

Article Title: Fungal chitin-binding glycoprotein induces Dectin-2-mediated allergic airway inflammation synergistically with chitin

doi: 10.1371/journal.ppat.1011878

Figure Lengend Snippet: (A) Bone marrow-derived dendritic cells (BMDCs) from C57BL/6 mice were incubated with recombinant LdpA (rLdpA) (1 μg/mL), chitin (50 μg/mL), rLdpA–chitin complex (rLdpA, 1 μg/mL; chitin, 50 μg/mL), lipopolysaccharide (LPS) (100 ng/mL), and phosphate-buffered saline (PBS) for 24 h. MHC class II, CD80, CD86, and CD40 cell-surface expression were measured by flow cytometry. (B) Proinflammatory cytokine and chemokine mRNA expression. After BMDCs derived from C57BL/6 mice were incubated with rLdpA (25 μg/mL), chitin (250 μg/mL), rLdpA–chitin complex (rLdpA, 1 μg/mL; chitin, 50 μg/mL), and PBS for 1.5 h, mRNA expression levels of Tnf-α , Il-1α , Il-1β , Il-12 p35 , Il-12 p40 , Il-6 , Kc/Cxcl1 , and Mip-2/Cxcl2 were measured by quantitative real-time PCR. mRNA expression levels were normalized relative to Gapdh mRNA levels and are shown as fold change relative to the control PBS group. (C, D) BMDCs derived from C57BL/6 mice were incubated with rLdpA (1 μg/mL), chitin (50 μg/mL), rLdpA–chitin complex (rLdpA, 1 μg/mL; chitin, 50 μg/mL), and PBS for 24 h, TNF-α, IL-1α, and KC/CXCL1 protein levels in the culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA) (C). Cytotoxicity was assessed by measuring the release of lactate dehydrogenase (LDH) (D). (E, F, G) BMDCs derived from C57BL/6, Card9 −/− , Myd88 −/− (E), Mincle −/− , MCL −/− (F), Dectin-2 −/− and Dectin-1 −/− (G) mice were incubated with rLdpA–chitin complex (rLdpA, 1 μg/mL; chitin, 50 μg/mL) and PBS for 24 h, and TNF-α, IL-1α, and KC/CXCL1 protein levels in the culture supernatant were measured by ELISA. Data are shown as the mean ± standard deviation (SD). * P < 0.05, ** P < 0.01, and *** P < 0.001 by one-way ANOVA with post hoc Tukey–Kramer test (B, C, D).

Article Snippet: Cells were stained with a mixture of fluorochrome-conjugated monoclonal antibodies (mAbs) to CD11c (N418; eBioscience), MHC class II (M5/114.15.2; Miltenyi Biotec), CD40 (FGK45.5; Miltenyi Biotec), CD80 (16-10A1; Miltenyi Biotec), and CD86 (PO3.3; Miltenyi Biotec) in FACS buffer.

Techniques: Derivative Assay, Incubation, Recombinant, Saline, Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction, Control, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in immunology

Article Title: Interleukins 4 and 21 Protect Anti-IgM Induced Cell Death in Ramos B Cells: Implication for Autoimmune Diseases.

doi: 10.3389/fimmu.2022.919854

Figure Lengend Snippet: FIGURE 9 | IL-21 rescued CD40L and anti-IgM induced cell death in human primary B cells. (A) Representative graphs of CFSE labelling cells showed no differentiation was induced by any stimulatory factors in 3 days. (B) Representative graphs showed that IL-21 could suppress cell death induced by CD40L and anti- IgM in terms of increased number of DAPI- cells. (C) Representative graphs showed that IL-21 was able to induce CD38+ B cell differentiation both in the absence and presence of CD40L.

Article Snippet: Cells were then seeded into 96-well round bottom plate at the density of 2E5 cells/100μl, and then challenged with 50 ng/ml IL-21 and 1 μg/ml CD40L (#6420-CL/CF, R&D Systems, Minneapolis, USA) or 2 μg/ml anti-IgM.

Techniques: Cell Differentiation

Journal: Science Advances

Article Title: High-throughput functional screening for next-generation cancer immunotherapy using droplet-based microfluidics

doi: 10.1126/sciadv.abe3839

Figure Lengend Snippet: HEK293FT cells were infected with a lentivirus antibody library and individually coencapsulated with Jurkat/NF-κB-GFP-hCD40 reporter cells and fluorescence-labeled secondary antibodies in droplets. Droplets containing reporter cells activated by antibodies secreted by the coencapsulated antibody–expressing cells were sorted. The sorted cells were expanded for the second round of selection, and enriched antibodies were identified by next-generation sequencing. ( A ) Schematic of possible time traces. ( B ) Proportions of different types of droplets for each round of selection were analyzed. ( C ) Bright-field and fluorescence images of the sorted droplets after the second round of selection. ( D ) Bar plot for the top 20 scFv clusters and their frequencies during the selection process. ( E ) The change in frequencies of the selected antibodies during the selection process. ( F ) Agonist activity of the selected antibodies was determined using the CD40 reporter cell line in the presence or absence of the cross-linking secondary antibody.

Article Snippet: Human CD40 (ACROBiosystems), GITR (ACROBiosystems), OX40 (ACROBiosystems), 4-1BB (ACROBiosystems), or bovine serum albumin (BSA) (Solarbio) was plated onto a microtiter plate at 4°C overnight.

Techniques: Infection, Fluorescence, Labeling, Expressing, Selection, Next-Generation Sequencing, Activity Assay

Journal: Science Advances

Article Title: High-throughput functional screening for next-generation cancer immunotherapy using droplet-based microfluidics

doi: 10.1126/sciadv.abe3839

Figure Lengend Snippet: ( A ) The FcγRIIB dependency of antibody C04. Jurkat/NF-κB-GFP-hCD40 reporter cells were stimulated with C04 antibody or anti-HEL antibody in coculture with FcγRIIB-overexpressing HEK293FT cells. Activation of the reporter cell line was analyzed by flow cytometry. ( B ) Activation of DCs or B cells by C04. DCs or B cells isolated from a donor were stimulated by C04 in the presence (left) or absence (right) of the cross-linking secondary antibody. Expression of CD86 was analyzed by flow cytometry. ( C ) OVA-specific CD8 + T cell response induced by C04 in FcγR/CD40-humanized mice. Transgenic mice were adoptively transferred with OVA-specific OT-I cells and treated with DEC-OVA, together with C04 or isotype control antibody. Mice were euthanized for the analysis of T cells. Each circle represents an individual mouse. ( D ) Antitumor effect of C04 in the syngeneic mouse model. FcγR/CD40-humanized mice were subcutaneously engrafted with MC38 tumor cells. When MC38 tumors were established (~100 mm 3 ), mice were treated with C04, CP-870,893, or isotype control antibody. Tumor volume and body weight were measured every 3 days until the end of the experiment. Data are represented as the means ± SEM. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Human CD40 (ACROBiosystems), GITR (ACROBiosystems), OX40 (ACROBiosystems), 4-1BB (ACROBiosystems), or bovine serum albumin (BSA) (Solarbio) was plated onto a microtiter plate at 4°C overnight.

Techniques: Activation Assay, Flow Cytometry, Isolation, Expressing, Transgenic Assay, Control

Journal: iScience

Article Title: Reduced mitochondrial respiration in T cells of patients with major depressive disorder

doi: 10.1016/j.isci.2021.103312

Figure Lengend Snippet:

Article Snippet: CD40 Antibody, anti-human, pure-functional grade (HB14) , Miltenyi Biotec , Cat# 130-094-133; RRID: AB_10839704.

Techniques: Recombinant, cDNA Synthesis, Gene Expression, Staining, Conjugation Assay, Software

Journal: Frontiers in Immunology

Article Title: Functional Plasticity of Gamma Delta T Cells and Breast Tumor Targets in Hypoxia

doi: 10.3389/fimmu.2018.01367

Figure Lengend Snippet: Natural killer group 2, member D (NKG2D) on gamma delta T cells (γδTcs) and MHC class I polypeptide-related sequence A (MICA)/B on breast cancer cell lines mediate γδTc cytotoxicity. Flow cytometric analysis of (A) MCF-7 ( n = 4) and (B) T47D ( n = 2) confirms that both cell lines express MICA/B. (C) Cytotoxicity assays in which NKG2D on γδTcs or MICA/B on MCF-7 cells are blocked with antibodies confirm γδTc recognition of breast cancer targets via this receptor/ligand interaction ( n = 3, representative of three independent experiments with three different donor cultures). (D) Blocking assays as in (C) using T47D targets ( n = 3). (E) Blocking macrophage inflammatory protein 1α (MIP1α), CCL5/regulated on activation, normal T cell expressed and secreted (RANTES), and CD40L/TNFSF5 does not decrease lysis of MCF-7 ( n = 3 independent experiments with two different donor cultures) or (F) T47D ( n = 2). Statistical analyses for (C–F) : one-way ANOVA, *** P < 0.001, **** P < 0.0001.

Article Snippet: The following ELISA kits were used: ELISA MAX Deluxe regulated on activation, normal T cell expressed and secreted (RANTES/CCL5) (BioLegend), Human macrophage inflammatory protein 1α (MIP1α) and Human CD40L Quantikine ELISA kits (R&D Systems), and Human MICA ELISA Kit (abcam).

Techniques: Sequencing, Blocking Assay, Activation Assay, Lysis

Journal: Frontiers in Immunology

Article Title: Functional Plasticity of Gamma Delta T Cells and Breast Tumor Targets in Hypoxia

doi: 10.3389/fimmu.2018.01367

Figure Lengend Snippet: Hypoxia induces secretion of macrophage inflammatory protein 1α (MIP1α), CCL5/regulated on activation, normal T cell expressed and secreted (RANTES), and CD40L/TNFSF5 by gamma delta T cells (γδTcs). (A) Culture supernatants from γδTc subjected to 40 h at 20 or 1% O 2 were analyzed by cytokine array. Cumulative results of three independent experiments for a panel of cytokines that were differentially secreted by γδTcs under hypoxia compared to normoxia are shown. Error bars are SEM; A.U. = arbitrary units; (B) ELISA validation of RANTES cytokine results shown in (A) for three independent experiments carried out at 20 and 1% O 2 for 40 h; (C) RANTES ELISA for eight hypoxia experiments carried out for 48 h at 20 and 2% O 2 ; (D) MIP1α ELISA for the same experiments shown in (B) ; (E) CD40L ELISA for culture 6A-16 subject to 48 h 20 or 2% O 2 , and two of the experiments shown in (B,D) . Statistical analyses for (A–E) : two-way ANOVA, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; (F–H) Western blot analysis of lysates from γδTc cultures subject to 20, 2, and/or 1% O 2 for 48 h as indicated. γδTc culture identification is given above the blots and molecular weight (MW) markers are shown on the left; corresponding β-actin loading controls are shown in the bottom panels; relative band intensities were quantified and are indicated in arbitrary units; (F) three examples shown for detection of hypoxia inducible factor 1-alpha (HIF1α) ( n = 6, 5 different donors) and CD40L ( n = 8, 7 γδTc cultures from six donors); (G) MIP1α ( n = 7, 6 γδTc cultures from five donors); (H) RANTES ( n = 7); and (I) induction of proteins in (F – H) was determined by dividing protein band intensities from hypoxic samples by their corresponding normoxia control, and averaging these values. Error bars are SD.

Article Snippet: The following ELISA kits were used: ELISA MAX Deluxe regulated on activation, normal T cell expressed and secreted (RANTES/CCL5) (BioLegend), Human macrophage inflammatory protein 1α (MIP1α) and Human CD40L Quantikine ELISA kits (R&D Systems), and Human MICA ELISA Kit (abcam).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Western Blot, Molecular Weight, Control