anti human cd55 bric216  (Bio-Rad)

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

Journal: The Journal of Immunology Author Choice

Article Title: The Influence of Human IgG Subclass and Allotype on Complement Activation

doi: 10.4049/jimmunol.2300307

Figure Lengend Snippet: Complement activation of IgG1 and IgG3 is independent of complement regulators. Complement-dependent cytotoxicity (CDC) activity of IGHG1*03 (black), IGHG3*01 (long hinge, red), and IGHG3*04 (short hinge, pink) specific to trinitrophenyl (TNP) on Raji and Ramos B cells including CD46, CD55, and CD59 knockout cell lines at an Ag density of 0.5 mM (medium) TNBS. Negative controls are shown as dotted line for TNP unlabeled cells, and a dashed line represents TNPylated cells in the absence of Abs. Data represent mean ± SEM of n = 3.

Article Snippet: To study the expression of different complement regulators, conjugated Abs were tested at the following dilutions: mouse anti-human CD46-3–FITC (1:229; in-house mAb), mouse anti-human CD55-allophycocyanin (1:250; clone IA10; BD Biosciences), mouse anti-human CD59-FITC (1:100; clone MEM-443; Thermo Fisher Scientific), rabbit anti-human complement receptor 1 (CR1; 1:500; in-house mAb) in combination with goat anti–rabbit IgG Alexa Fluor 488 (1:500; clone A11034; Invitrogen) and mouse anti–human CR2-BV605 (1:100; clone BB-ly4; BD Biosciences).

Techniques: Activation Assay, Activity Assay, Knock-Out

Journal: Cell Reports Methods

Article Title: Sequence enrichment profiles enable target-agnostic antibody generation for a broad range of antigens

doi: 10.1016/j.crmeth.2023.100475

Figure Lengend Snippet:

Article Snippet: PE Mouse anti-Human CD55 , BD Biosciences , Cat# 561901; RRID: AB_10893598.

Techniques: Produced, Recombinant, Sequencing, Software

Journal: mBio

Article Title: CD55 Facilitates Immune Evasion by Borrelia crocidurae , an Agent of Relapsing Fever

doi: 10.1128/mbio.01161-22

Figure Lengend Snippet: Screening the BASEHIT human exoproteome library to identify host proteins that interact with Borrelia strains that cause relapsing fever. (A) Schematic of yeast display screen: the BASEHIT yeast library, displaying 1,031 human proteins on the yeast surface, was mixed with surface-biotinylated Borrelia isolates. Each human protein is encoded by a unique bar-coded plasmid. Yeast cells binding to Borrelia spirochetes were isolated by magnetic separation using streptavidin microbeads, and next-generation sequencing was used to identify human proteins. (B) Host interactions with B. crocidurae or B. persica grown at 33°C. Each symbol represents one human protein. CD55 is represented by a larger, solid square or circle. The list of proteins that bound to B. crocidurae and/or B. persica is shown in Table S1 . The score for each protein is defined as the overall enrichment for that corresponding gene (relative to the unselected library) multiplied by the percentage of barcodes associated with the enriched gene (defined as a log fold change [logFC] of >0). The dotted line reflects a BASEHIT score of 1, and all genes that had higher scores are listed in Table S1. (C) CD55 interactions with different Borrelia species. Samples from 29 Borrelia isolates were screened against the host BASEHIT protein library. The y axis represents the CD55 score as the overall enrichment for CD55 of each isolate (relative to the unselected library) multiplied by the percentage of barcodes associated with the enriched CD55 (defined as logFC of >0).

Article Snippet: The binding of B. crocidurae to human CD55-Fc was measured using an anti-human CD55 mouse monoclonal antibody (MAB2009; R&D systems) and a goat anti-mouse IgG (H+L) Alexa Fluor 488- conjugated secondary antibody (ThermoFisher Scientific) (1:1,000).

Techniques: Plasmid Preparation, Binding Assay, Isolation, Next-Generation Sequencing

Journal: mBio

Article Title: CD55 Facilitates Immune Evasion by Borrelia crocidurae , an Agent of Relapsing Fever

doi: 10.1128/mbio.01161-22

Figure Lengend Snippet: CD55 binds B. crocidurae and B. persica . (A and B) Binding of human and mouse CD55 to B. crocidurae (A) and B. persica (B). (A) B. crocidurae cultures were grown to a density of 10 5 CFU/mL and incubated with recombinant CD55-His 8 (50 μg/mL). PGLYRP1 has previously been shown to bind spirochetes that cause Lyme borreliosis and relapsing fever and was used as a positive control. B. crocidurae ’s binding to recombinant protein was measured by flow cytometry using a secondary Alexa Fluor 488 (AF488)-conjugated anti-His 6 monoclonal antibody. Overlay histograms show protein binding to B. crocidurae spirochetes identified using the secondary antibody. Binding of recombinant tick protein IsPDIA3-His 8 (50 μg/mL) to B. crocidurae was used as a negative control. Background binding of AF488-conjugated anti-His 6 monoclonal antibody alone with B. crocidurae is shown by the gray-shaded region. Results from one of two representative experiments are shown here. (B) B. persica cultures were grown to a density of 10 5 CFU/mL and incubated with recombinant CD55-His 8 (50 μg/mL). B. persica ’s binding to recombinant CD55-His 8 was measured using a secondary AF488-conjugated anti-His 6 monoclonal antibody.

Article Snippet: The binding of B. crocidurae to human CD55-Fc was measured using an anti-human CD55 mouse monoclonal antibody (MAB2009; R&D systems) and a goat anti-mouse IgG (H+L) Alexa Fluor 488- conjugated secondary antibody (ThermoFisher Scientific) (1:1,000).

Techniques: Binding Assay, Incubation, Recombinant, Positive Control, Flow Cytometry, Protein Binding, Negative Control

Journal: mBio

Article Title: CD55 Facilitates Immune Evasion by Borrelia crocidurae , an Agent of Relapsing Fever

doi: 10.1128/mbio.01161-22

Figure Lengend Snippet: CD55 binding to B. crocidurae and its impact on complement inhibition. (A and B) Binding of human and mouse CD55 to B. crocidurae . (A) B. crocidurae cultures were grown to a density of 10 5 CFU/mL and incubated with recombinant human CD55-Fc (50 μg/mL) alone or in the presence of mouse CD55-His 8 (100 μg/mL). B. crocidurae ’s binding to human CD55-Fc was measured by flow cytometry using a human CD55 monoclonal antibody (MAB2009; R&D systems) and goat anti-mouse Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific) (1:1,000). The results from one of five representative experiments are shown here. (B) Data from five independent experiments are shown here. The y axis represents arbitrary fluorescence units (mean fluorescence intensity of AF488) from the B. crocidurae spirochete population. Data were acquired on a BD LSRII flow cytometer and analyzed by FlowJo software. The bars represent mean values ± standard deviations (SD), and P values were determined by the Student t test (Mann-Whitney test of the data for B. crocidurae incubated with hCD55 Fc versus mCD55/hCD55Fc [ P = 0.0079]). (C and D) Binding of mouse CD55 to proteinase K-treated or untreated B. crocidurae spirochetes. (C) B. crocidurae cultures were grown to a density of 10 5 CFU/mL and incubated in the presence or absence of proteinase K (0.2 mg/mL) at 37°C for 10 min. Subsequently, the proteinase K activity was quenched using a Roche cOmplete proteinase inhibitor cocktail and spirochetes were washed with PBS thrice. Borrelia spirochetes were incubated with recombinant mouse CD55-His 8 (50 μg/mL). B. crocidurae ’s binding to mouse CD55 was measured by flow cytometry using a secondary AF488-conjugated anti-His 6 monoclonal antibody. Background binding of AF488-conjugated anti-His 6 monoclonal antibody alone with B. crocidurae is shown by the gray-shaded region. The results from one of five representative experiments are shown here. (D) Data from five independent experiments are plotted here. The y axis represents the mean fluorescence intensities of AF488 from B. crocidurae . Statistical significance was assessed using the nonparametric Student t test (Mann-Whitney test of the data for proteinase K-pretreated B. crocidurae incubated with mCD55 versus untreated B. crocidurae incubated with mCD55 [ P = 0.0079]). (E) Complement-mediated killing of B. crocidurae in the presence or absence of recombinant human CD55. Human CD55 (100 μg/mL) was incubated with B. crocidurae for 2 h in the presence or absence of immune serum from mice that were infected 30 days previously with B. crocidurae . Viability was assessed by observing spirochete movement under dark-field microscopy. The growth inhibition of B. crocidurae was calculated based on the growth of untreated B. crocidurae . The bars represent mean values ± SD, and P values were determined by the Student t test (Mann-Whitney test of the data for 20% serum versus 20% serum plus hCD55 [ P = 0.0023] or 40% serum versus 40% serum plus hCD55 [ P = 0.0023]). Data from seven independent experiments are shown here. (F) Complement-mediated killing of B. crocidurae in the presence or absence of recombinant mouse or human CD55. Human or mouse CD55-His 8 (100 μg/mL) was incubated with B. crocidurae for 24 h in the presence or absence of 20% serum from mice that were infected 30 days previously with B. crocidurae . Viability was assessed using the Bac-Titer Glo assay. The growth inhibition of B. crocidurae was calculated based on the growth of B. crocidurae incubated with PBS alone. Statistical significance was assessed using the Student t test (PBS versus mCD55 [ P = 0.0132] or PBS versus hCD55 [ P = 0.0105]). Data from four independent experiments performed in triplicates are shown here.

Article Snippet: The binding of B. crocidurae to human CD55-Fc was measured using an anti-human CD55 mouse monoclonal antibody (MAB2009; R&D systems) and a goat anti-mouse IgG (H+L) Alexa Fluor 488- conjugated secondary antibody (ThermoFisher Scientific) (1:1,000).

Techniques: Binding Assay, Inhibition, Incubation, Recombinant, Flow Cytometry, Fluorescence, Software, MANN-WHITNEY, Activity Assay, Infection, Microscopy, Glo Assay

Journal: mBio

Article Title: CD55 Facilitates Immune Evasion by Borrelia crocidurae , an Agent of Relapsing Fever

doi: 10.1128/mbio.01161-22

Figure Lengend Snippet: B. crocidurae -induced rosette formation is inhibited by CD55. (A) B. crocidurae interactions with erythrocytes from WT and CD55 KO mice were compared by microscopy. Erythrocytes from WT and CD55 KO mice were labeled with the fluorescent dye PKH67. The labeled RBCs were incubated with 10 5 B. crocidurae spirochetes in a 96-well, flat-bottom plate at 37°C. The resulting rosettes and aggregates were observed by fluorescence microscopy, and aggregate sizes were measured ( Fig. S4 ). (B) B. crocidurae interactions with erythrocytes from WT and CD55 KO mice were compared. Erythrocytes from WT and CD55 KO mice were incubated with 10 6 B. crocidurae spirochetes in 0.2-mL PCR tubes at 37°C as described before . Hemoglobin from erythrocytes that interacted with B. crocidurae was quantified using the QuantiChrom hemoglobin assay kit. Data from three independent experiments performed in triplicates are presented. (C) B. crocidurae ’s interaction with human RBCs in the presence of anti-CD55 neutralizing antibody (R&D Systems) was measured. Human RBCs were preincubated with either anti-human CD55 neutralizing antibody or another antibody (anti-CD3 antibody) as an isotype control for 20 min. The RBCs were incubated with 10 6 B. crocidurae spirochetes. The interactions between human RBCs and B. crocidurae spirochetes were measured by flow cytometry. Data from three independent experiments are presented. (D) B. crocidurae ’s interactions with RBCs in the presence of recombinant CD55 or PGLYRP1 were compared. RBCs from C57BL/6 mice were incubated with 10 6 B. crocidurae spirochetes at 37°C in the presence or absence of recombinant human (h) or mouse (m) CD55 (100 μg/mL). Hemoglobin from RBCs that interacted with B. crocidurae was quantified.

Article Snippet: The binding of B. crocidurae to human CD55-Fc was measured using an anti-human CD55 mouse monoclonal antibody (MAB2009; R&D systems) and a goat anti-mouse IgG (H+L) Alexa Fluor 488- conjugated secondary antibody (ThermoFisher Scientific) (1:1,000).

Techniques: Microscopy, Labeling, Incubation, Fluorescence, Control, Flow Cytometry, Recombinant

Journal: mBio

Article Title: CD55 Facilitates Immune Evasion by Borrelia crocidurae , an Agent of Relapsing Fever

doi: 10.1128/mbio.01161-22

Figure Lengend Snippet: Borrelia burdens and cytokine profiles in CD55 KO mice. (A) B. crocidurae spirochetes were inoculated into WT (C57BL/6) or CD55 KO mice to assess spirochete infection and immune response. The burdens of Borrelia spirochetes in murine blood at 1 and 2 days after infection were determined. The spleen size was determined at 10 days postinfection. (B) Wild-type C57BL/6 and CD55 knockout (KO) mice ( n = 6 per group minimum) were infected with 10 5 B. crocidurae spirochetes by intraperitoneal injection. The B. crocidurae burdens in the blood at 2 days postinfection were assessed by qPCR using the Borrelia- specific flagellin subgroup B gene ( flaB ) normalized to mouse β-actin . The results from three independent experiments are shown here. (C) The extent of splenomegaly was expressed as splenic weights at 10 days postinfection in Wt and CD55KO mice infected with B. crocidurae . Each data point represents the value for an individual animal. The bars represent mean values ± SD, and P values were determined by the Student t test.

Article Snippet: The binding of B. crocidurae to human CD55-Fc was measured using an anti-human CD55 mouse monoclonal antibody (MAB2009; R&D systems) and a goat anti-mouse IgG (H+L) Alexa Fluor 488- conjugated secondary antibody (ThermoFisher Scientific) (1:1,000).

Techniques: Infection, Knock-Out, Injection

Journal: mBio

Article Title: CD55 Facilitates Immune Evasion by Borrelia crocidurae , an Agent of Relapsing Fever

doi: 10.1128/mbio.01161-22

Figure Lengend Snippet: Serological response in CD55 KO mice following B. crocidurae infection. B. crocidurae spirochetes were inoculated into WT (C57BL/6) or CD55 KO mice, and serum cytokine profiles were assessed in both wild-type and CD55 KO mice at day 2 and day 4 postinfection ( n = 7 in each group) using a mouse cytokine/chemokine 31-plex (MD-31) array. (A to E) Increases in proinflammatory cytokines IL-6 (A), IL-1α (B), and TNF-α (C) and chemokines RANTES/CCL5 (D) and MIP1α/CCL3 (E) were observed in infected CD55 KO mice compared to the levels in parent C57BL/6 mice. Each data point represents the result for an individual animal in the corresponding group. The bars represent mean values ± SD, and P values were determined by the Student t test. (F) C5a levels in wild-type C57BL/6 (WT) and CD55 KO mice. Four days postinfection, C5a levels were measured by ELISA using serum from infected WT or infected CD55 KO mice. Serum from uninfected mice was used for comparison. All sera were used at a 1:1,000 dilution. Each data point represents the result for an individual animal in the corresponding group. The bars represent mean values ± SD, and P values were determined by the Student t test.

Article Snippet: The binding of B. crocidurae to human CD55-Fc was measured using an anti-human CD55 mouse monoclonal antibody (MAB2009; R&D systems) and a goat anti-mouse IgG (H+L) Alexa Fluor 488- conjugated secondary antibody (ThermoFisher Scientific) (1:1,000).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Comparison

Journal: mBio

Article Title: CD55 Facilitates Immune Evasion by Borrelia crocidurae , an Agent of Relapsing Fever

doi: 10.1128/mbio.01161-22

Figure Lengend Snippet: Gene expression analysis by RNA sequencing reveals key genetic signatures following B. crocidurae infection. (A) Transcriptome analysis was done with WT (C57BL/6) or CD55 KO mice to assess genetic signatures following relapsing fever infection. Mice were infected with 10 5 B. crocidurae spirochetes, and blood was collected at 2 dpi. The results for CD55 KO and C57BL/6 WT mice were compared. (B) Principal-component analysis (PCA) of transcriptome data from murine whole-blood samples. Each dot represents the data for one mouse. KOIN, B. crocidurae -infected CD55 KO mice; WTIN, B. crocidurae -infected C57BL/6 WT mice. (C) Differentially expressed genes. The x axis shows the log 2 -transformed fold changes in CD55 KO mice relative to the expression levels in WT mice at 2 days postinfection with B. crocidurae . (D) Heat map of the expression of selected genes in KOIN and WTIN. (E) Signaling pathways involved in the response to B. crocidurae infection in KOIN and WTIN were identified by KEGG pathway enrichment analysis. The key immune pathways enriched included T cell receptor and B cell receptor signaling, natural killer cell-mediated cytotoxicity, NF-κB signaling, antigen processing and presentation, and primary immunodeficiency. (F) B. crocidurae -specific IgM levels in uninfected wild-type C57BL/6 (WT) and CD55 KO mice were compared with those in the infected animals at two time points (12 days and 21 days postinfection). Whole-cell lysate of B. crocidurae was used to coat the wells of a microtiter plate, and serum from uninfected WT, infected WT, uninfected CD55 KO, or infected CD55 KO mice was used at a 1:200 dilution. The binding was measured using a secondary HRP-conjugated anti-mouse IgM. Each data point represents the result for an individual animal in the corresponding group. The bars represent mean values ± SD, and P values were determined using the Student t test.

Article Snippet: The binding of B. crocidurae to human CD55-Fc was measured using an anti-human CD55 mouse monoclonal antibody (MAB2009; R&D systems) and a goat anti-mouse IgG (H+L) Alexa Fluor 488- conjugated secondary antibody (ThermoFisher Scientific) (1:1,000).

Techniques: Gene Expression, RNA Sequencing, Infection, Transformation Assay, Expressing, Protein-Protein interactions, Binding Assay