Journal: Bioactive Materials

Article Title: Upconversion nanoparticles regulated drug & gas dual-effective nanoplatform for the targeting cooperated therapy of thrombus and anticoagulation

doi: 10.1016/j.bioactmat.2022.03.013

Figure Lengend Snippet: NIR responsive characteristics of UCATS. A) UCL spectra of UCM, UCA and UCATS. B) UV–vis absorption spectra changes of UCA before and after irradiation with 808 nm NIR light (2 W cm −2 ). C) UV–vis absorption spectra changes of SNAP before and after irradiation by vis (room light) and UV (365 nm) light for different time. D) NIR-triggered controllable release of UK from UCATS-UK. E) NIR-triggered NO release from UCATS at different concentrations and SNAP. 808 nm NIR: 2 W cm −2 . F) On/Off behavior of NO release triggered by 808 nm NIR light (2 W cm −2 ). G) The fluorescent images of HUVECs stained with NO fluorescent probe (DAF-FM DA) and Hoechst 33342 after being treated with UCATS under dark conditions or 808 nm NIR irradiation (1 W cm −2 ). Scale bar = 50 μm. Data are means ± s.d. (n = 3).

Article Snippet: Ammonium fluoride (NH 4 F, 98.0%) and tetraethyl orthosilicate (TEOS, 99.0%) were purchased from Alfa Co., LTD. DAF-FM DA and Hoechst 33342 were purchased from MCE Co., LTD. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, 97.0%) was purchased from J&K Scientific.

Techniques: Irradiation, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Epidermal growth factor receptor targeting IgG3 triggers complement-mediated lysis of decay-accelerating factor expressing tumor cells through the alternative pathway amplification loop.

doi: 10.4049/jimmunol.1400329

Figure Lengend Snippet: FIGURE 4. CML triggered by anti–EGFR-IgG3 negatively correlates with CD55 and CD59 expression levels. (A) Surface expression levels of CD46, CD55, and CD59 on analyzed cell lines were quantified by calibrated flow cytometry. Means 6 SEM of at least three independent experiments are presented. (B) Correlations between CD46, CD55, or CD59 and anti–EGFR-IgG3–mediated CDC were calculated for all four cell lines. CDC results at 2 mg/ml Ab concentration were taken from experiments presented in Fig. 2. (C) A431 cells were seeded into 10-cm plates and grown overnight. On the following day, cells were transfected with 50 nM control siRNA or with single siRNAs specific for CD46, CD55, CD59, or with a combination of all three mCRP-specific siRNAs for 72 h. Efficiency of siRNA-induced knockdown was analyzed by direct flow cytometry using fluorochrome-labeled, mCRP- specific Abs (CD46-Pacific blue, CD55-PE, CD59-FITC), or respective control Abs. (D–G) CDC against control siRNA or mCRP-specific, siRNA- transfected A431 cells was analyzed by 3-h [51Cr] release assays in the presence of 25% v/v human serum and anti–EGFR-IgG1 (upper panels), anti– EGFR-IgG3 (lower panels), as well as the respective control Abs at increasing concentrations. (H) Concentration-dependent binding of CD55-Ab (BRIC216, mouse IgG1) to A431 cells was analyzed by indirect immunofluorescence. Results from one representative experiment are presented. (I) CDC triggered by anti–EGFR-IgG1, anti–EGFR-IgG3, or respective control Abs (all at 66.67 nM) against A431 cells in the presence of saturating concentrations of CD55-Ab or a control Ab (both at 66.67 nM) was analyzed by 3-h [51Cr] release assays in the presence of 25% v/v human (Figure legend continues)

Article Snippet: To block complement regulatory activity of CD55, we used mouse anti-human CD55 (66.67 nM, BRIC216, mouse IgG1; Bio-Rad) blocking mAb in CDC experiments at saturating concentrations.

Techniques: Expressing, Cytometry, Concentration Assay, Transfection, Control, Knockdown, Labeling, Binding Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Epidermal growth factor receptor targeting IgG3 triggers complement-mediated lysis of decay-accelerating factor expressing tumor cells through the alternative pathway amplification loop.

doi: 10.4049/jimmunol.1400329

Figure Lengend Snippet: FIGURE 5. CD55 dampens anti–EGFR-IgG3–triggered CML and promotes C1q-dependent induction of AP amplification. BHK-EGFR+ #5 cells were transiently transfected with a control vector or a CD55 vector for 48 h. (A) Cell-surface expression of CD55 was analyzed by direct flow cytometry using PE-conjugated CD55-specific or control Abs. (B–D) The influence of CD55 overexpression on anti–EGFR-IgG3–mediated CDC was investigated by [51Cr] release assays either (B) in an Ab concentration–response curve, (C) in a time-dependent manner, or (D) in serum titration experiments. (E–H) The influence of the alternative complement pathway inhibitor CRIg (E and G), the presence of C1q in serum (F; at 66.67 nM Ab concentration; mean 6 SEM of triplicates), as well as of factor B (H; 13.33 nM Ab concentration, 12.5% v/v factor B–depleted serum, 200 mg/ml factor B), on anti–EGFR-IgG3–mediated CDC was analyzed using either (E and F) control vector–transfected or CD55 vector–transfected BHK-EGFR+ #5 cells or (G and H) DiFi cells (66.67 nM Ab concentration). (I) Deposition of factor Bb on control vector– or CD55 vector–transfected BHK-EGFR+ #5 cells was analyzed by flow cytometry. Relative deposition levels were calculated by equating RFI measured in the absence of Ab with 100%. Results are presented as mean 6 SEM of at least three independent experiments with different blood donors. *p # 0.05 anti–EGFR-IgG3 versus respective control Ab; (B–D, I) #p # 0.05 control vector versus CD55 vector; (E and G) #p # 0.05 without CRIg-Fc versus CRIg-Fc; (H) #p # 0.05 w/o factor B versus with factor B.

Article Snippet: To block complement regulatory activity of CD55, we used mouse anti-human CD55 (66.67 nM, BRIC216, mouse IgG1; Bio-Rad) blocking mAb in CDC experiments at saturating concentrations.

Techniques: Transfection, Control, Plasmid Preparation, Expressing, Cytometry, Over Expression, Concentration Assay, Titration

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Epidermal growth factor receptor targeting IgG3 triggers complement-mediated lysis of decay-accelerating factor expressing tumor cells through the alternative pathway amplification loop.

doi: 10.4049/jimmunol.1400329

Figure Lengend Snippet: FIGURE 6. Overview of complement activation by human anti–EGFR-IgG3 in the context of CD55 expression. On CD55-deficient target cells (left panel), anti–EGFR-IgG3 mediates strong C3b but low C4b deposition and induces assembly of classical and alternative C3 convertases, predominantly resulting in the induction of fast and efficient CDC via the classical pathway of complement activation. In contrast, on CD55-expressing target cells (right panel), CD55 mainly accelerates the decay of low amounts of classical C3 convertases, leading to amplification of the AP and finally to slow and inefficient CDC induction.

Article Snippet: To block complement regulatory activity of CD55, we used mouse anti-human CD55 (66.67 nM, BRIC216, mouse IgG1; Bio-Rad) blocking mAb in CDC experiments at saturating concentrations.

Techniques: Activation Assay, Expressing

Journal: iScience

Article Title: CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition

doi: 10.1016/j.isci.2024.110120

Figure Lengend Snippet: Receptor-ligand CRISPR-Cas9 activation screen reveals that CD55 interacts with HLA-C∗07:01-VRIG tetramers (A) Schematic of the receptor ligand CRISPR-Cas9 activation screen. K562 cells transduced with a genome-wide activation library were stained with a pool of three HLA tetramers (HLA-A∗02:01-NLVP, HLA-B∗07:02-TPRV, and HLA-C∗07:01-VRIG), and enriched gRNAs in stained cells were identified using NGS. (B) SigmaFC scores of genes from two replicate screens. SigmaFC scores were calculated using PinAplPy, and top hits are annotated. (C) K562 cells stably expressing dCas9 and transduced with a gRNA upregulating CD55 or a control guide were stained with the HLA-A, -B, -C, or tetramers as in (A) or with HLA-E∗01:01-VMAP tetramers and analyzed by flow cytometry. (D) In vitro co-immunoprecipitation of recombinant CD55-Fc with HLA-A∗02:01-NLVP, HLA-B∗07:02-TPRV, HLA-C∗07:01-VRIG, or HLA-E∗01:01-VMAP tetramers. (E) Three different cell lines (HeLa, PC-3M, or SiHa) that express CD55 endogenously were stained for CD55 (top) or with HLA-C∗07:01-VRIG tetramers (bottom) and analyzed by flow cytometry. (F) HeLa wild-type or HeLa CD55 KO cells were stained with αCD55 or HLA-C∗07:01-VRIG tetramers and analyzed by flow cytometry. All data except (B) represent at least three independent experiments. CRISPRa, CRISPR activation screen; TMs, tetramers; WT, wild-type; KO, knockout. Related to Figure S1 and Table S1 .

Article Snippet: Antibodies used in this study were anti-heparan sulfate chains (AMSBIO, F58-10E4), FITC anti-human CD55 (Biolegend, 311306), anti-human CD55 BRIC110 (ARP, 08-9402-2, targets SCR2 of CD55), anti-human CD55 BRIC216 (Biorad, MCA914T, targets SCR3 of CD55), anti-human CD55 MAB2009 (R&D systems, MAB2009-SP, targets SCR1 of CD55), anti-SDC2 APC (R&D systems, FAB2965A), anti-SDC4 APC (R&D systems, FAB29181A), anti-CD55 APC (Biolegend, #311311), or goat-anti-mouse APC (Biolegend, #405308).

Techniques: CRISPR, Activation Assay, Transduction, Genome Wide, Staining, Stable Transfection, Expressing, Control, Flow Cytometry, In Vitro, Immunoprecipitation, Recombinant, Knock-Out

Journal: iScience

Article Title: CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition

doi: 10.1016/j.isci.2024.110120

Figure Lengend Snippet: Interaction of CD55 with HLA-C∗07:01-VRIG tetramers is allotype and peptide specific (A) HEK293T cells were transfected with a plasmid containing GFP and a truncation mutant of CD55 and analyzed by flow cytometry. GFP+ positive cells were analyzed for staining with HLA-C∗07:01-VRIG. Each mutant removes an additional SCR domain from CD55. Data are represented as mean ± SD. (B) HeLa cells were stained with HLA-C∗07:01-VRIG tetramers after pre-incubation with CD55 blocking antibodies targeting different SCR domains on CD55 and analyzed by flow cytometry. (C) HeLa cells were stained with either HLA-C∗07:01 or HLA-C∗07:02 tetramers loaded with the VRIG peptide and analyzed by flow cytometry. (D) HeLa cells were stained with HLA-C∗07:01 tetramers loaded with different alanine mutants of the VRIGHLYIL peptide and analyzed by flow cytometry. (E) CD55-Fc was immobilized on a Prot-G chip for SPR data using HLA-C∗07:01-VRIG tetramers as analyte to determine interaction on and off rates and K D . Response units were measured with increasing concentrations of HLA-C∗07:01-VRIG tetramers. All data represent at least three independent experiments, except (E), which represents a biological duplicate. FL, full length. Related to Figure S2 , Tables S2 and .

Article Snippet: Antibodies used in this study were anti-heparan sulfate chains (AMSBIO, F58-10E4), FITC anti-human CD55 (Biolegend, 311306), anti-human CD55 BRIC110 (ARP, 08-9402-2, targets SCR2 of CD55), anti-human CD55 BRIC216 (Biorad, MCA914T, targets SCR3 of CD55), anti-human CD55 MAB2009 (R&D systems, MAB2009-SP, targets SCR1 of CD55), anti-SDC2 APC (R&D systems, FAB2965A), anti-SDC4 APC (R&D systems, FAB29181A), anti-CD55 APC (Biolegend, #311311), or goat-anti-mouse APC (Biolegend, #405308).

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Flow Cytometry, Staining, Incubation, Blocking Assay

Journal: iScience

Article Title: CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition

doi: 10.1016/j.isci.2024.110120

Figure Lengend Snippet:

Article Snippet: Antibodies used in this study were anti-heparan sulfate chains (AMSBIO, F58-10E4), FITC anti-human CD55 (Biolegend, 311306), anti-human CD55 BRIC110 (ARP, 08-9402-2, targets SCR2 of CD55), anti-human CD55 BRIC216 (Biorad, MCA914T, targets SCR3 of CD55), anti-human CD55 MAB2009 (R&D systems, MAB2009-SP, targets SCR1 of CD55), anti-SDC2 APC (R&D systems, FAB2965A), anti-SDC4 APC (R&D systems, FAB29181A), anti-CD55 APC (Biolegend, #311311), or goat-anti-mouse APC (Biolegend, #405308).

Techniques: Virus, Recombinant, Blocking Assay, Genome Wide, Activation Assay, CRISPR, Knock-Out, Mutagenesis, Plasmid Preparation, Software, Imaging