Journal: Immunology

Article Title: Anti‐CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems

doi: 10.1111/imm.13437

Figure Lengend Snippet: Effect of anti‐CD52‐Ab treatment on the phenotype of periphery innate immune cells in EAE mice at first day post‐injection. Single‐cell suspensions were obtained from blood and spleen tissue of anti‐CD52‐Ab or vehicle‐treated EAE mice at first day after the final dose injection. Cells were stained for various surface markers and analysed by flow cytometry. (A) Flow cytometry gating strategy. (B) Blood and spleen CD11c + cells were analysed for expression of MHC‐II ( n = 9), CD40 ( n = 5), CD80 ( n = 5) and CD86 ( n = 9 blood; n = 5 spleen). (C) Blood and spleen CD11b + cells were analysed for expression of MHC‐II ( n = 8), CD40 ( n = 5), CD80 ( n = 5), CD86 ( n = 5), CD38 ( n = 5) and CD206 ( n = 9 blood; n = 7 spleen). Unpaired two‐tailed Student's t test. * p < 0·05, ** p < 0·01, *** p < 0·001

Article Snippet: To block non‐specific Fc receptors, cells were resuspended in anti‐mouse CD16/CD32 Fc block (eBioscience) for 10 min before incubation with the appropriate antibodies against: CD11b (#130‐113‐236), CD11c (#130‐102‐545), CD38 (#130‐109‐257), CD40 (#130‐105‐376 or #130‐102‐533), CD80 (#130‐116‐465), CD86 (#130‐105‐137), MHC‐II (#130‐107‐942), all were purchased from MACS Miltenyi Biotec; and CD45 (#11‐0451‐82) and CD206 (#12‐2061‐82), all were purchased from Thermo Fisher Scientific.

Techniques: Injection, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: Immunology

Article Title: Anti‐CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems

doi: 10.1111/imm.13437

Figure Lengend Snippet: Effect of anti‐CD52‐Ab treatment on the phenotype and function of CNS innate immune cells in EAE mice at first day post‐injection. Single‐cell suspensions were obtained from CNS (brain and spinal cord) tissue of anti‐CD52‐Ab or vehicle‐treated EAE mice at first day after the final treatment dose. Cells were stained for various surface markers and analysed by flow cytometry. (A) Flow cytometry gating strategy. (B) CD11b + CD45 lo/neg resident microglia and (C) CNS CD11b + CD45 hi infiltrating macrophages and were analysed for expression of MHC‐II ( n = 10), CD40 ( n = 5), CD80 ( n = 5), CD86 ( n = 5 microglia; n = 9 macrophages) and CD206 ( n = 7 microglia; n = 5 macrophages). Unpaired two‐tailed Student's t test. CD11b + cells were isolated from CNS tissue of anti‐CD52‐Ab or vehicle‐treated mice at first day after the final treatment dose. (D) Respiratory burst activity of CD11b + ‐isolated cells. (E) Phagocytosis of FITC‐coated beads by CD11b + isolated cells. Unpaired two‐tailed Student's t test. (F) Cells were left unstimulated or were stimulated with LPS or Poly(I:C)(PIC) for 24 h and cytokine production determined by ELISA. Two‐way ANOVA with Sidak multiple comparisons test, * p < 0·05, ** p < 0·01, *** p < 0·001

Article Snippet: To block non‐specific Fc receptors, cells were resuspended in anti‐mouse CD16/CD32 Fc block (eBioscience) for 10 min before incubation with the appropriate antibodies against: CD11b (#130‐113‐236), CD11c (#130‐102‐545), CD38 (#130‐109‐257), CD40 (#130‐105‐376 or #130‐102‐533), CD80 (#130‐116‐465), CD86 (#130‐105‐137), MHC‐II (#130‐107‐942), all were purchased from MACS Miltenyi Biotec; and CD45 (#11‐0451‐82) and CD206 (#12‐2061‐82), all were purchased from Thermo Fisher Scientific.

Techniques: Injection, Staining, Flow Cytometry, Expressing, Two Tailed Test, Isolation, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Immunology

Article Title: Anti‐CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems

doi: 10.1111/imm.13437

Figure Lengend Snippet: Effect of anti‐CD52‐Ab treatment on the phenotype of periphery and CNS innate immune cells in EAE mice at three weeks post‐injection. Single‐cell suspensions were obtained from blood, spleen and CNS (brain and spinal cord) tissue of anti‐CD52‐Ab or vehicle‐treated mice three weeks after the final treatment dose. Cells were stained for various surface markers and analysed by flow cytometry. (A) Blood and spleen CD11c + cells were analysed for expression of MHC‐II ( n = 9), CD40 ( n = 5), CD80 ( n = 5) and CD86 ( n = 9 blood; n = 5 spleen). (B) Blood and spleen CD11b + cells were analysed for expression of MHC‐II ( n = 8), CD40 ( n = 5), CD80 ( n = 5), CD86 ( n = 5), CD38 ( n = 5) and CD206 ( n = 9 blood; n = 7 spleen). (C) CNS CD11b + CD45 hi ‐infiltrating macrophages and CD11b + CD45 lo/neg resident microglia were analysed for expression of MHC‐II ( n = 10), CD40 ( n = 5), CD80 ( n = 5), CD86 ( n = 5 microglia; n = 9 macrophages) and CD206 ( n = 7 microglia; n = 5 macrophages). Unpaired two‐tailed Student's t test. * p < 0·05, ** p < 0·01, *** p < 0·001

Article Snippet: To block non‐specific Fc receptors, cells were resuspended in anti‐mouse CD16/CD32 Fc block (eBioscience) for 10 min before incubation with the appropriate antibodies against: CD11b (#130‐113‐236), CD11c (#130‐102‐545), CD38 (#130‐109‐257), CD40 (#130‐105‐376 or #130‐102‐533), CD80 (#130‐116‐465), CD86 (#130‐105‐137), MHC‐II (#130‐107‐942), all were purchased from MACS Miltenyi Biotec; and CD45 (#11‐0451‐82) and CD206 (#12‐2061‐82), all were purchased from Thermo Fisher Scientific.

Techniques: Injection, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: Immunology

Article Title: Anti‐CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems

doi: 10.1111/imm.13437

Figure Lengend Snippet: Effect of anti‐CD52‐Ab treatment on the function of periphery and CNS innate immune cells in SCID mice at first day post‐injection. SCID mice were treated with either vehicle or 10 mg/kg murine anti‐CD52‐Ab‐Ab for five consecutive days. Single‐cell suspensions were then obtained from blood and spleen tissues first day after the final treatment dose. Cells were stained for various surface markers and analysed by flow cytometry. (A) Blood and spleen CD11c + cells were analysed for expression of MHC‐II, CD40, CD80 and CD86 (all n = 7). (B) Blood and spleen CD11b + cells were analysed for expression of MHC‐II, CD40, CD80, CD86, and CD206 (all n = 7). Unpaired two‐tailed Student's t test. * p < 0·05, ** p < 0·01, *** p < 0·001

Article Snippet: To block non‐specific Fc receptors, cells were resuspended in anti‐mouse CD16/CD32 Fc block (eBioscience) for 10 min before incubation with the appropriate antibodies against: CD11b (#130‐113‐236), CD11c (#130‐102‐545), CD38 (#130‐109‐257), CD40 (#130‐105‐376 or #130‐102‐533), CD80 (#130‐116‐465), CD86 (#130‐105‐137), MHC‐II (#130‐107‐942), all were purchased from MACS Miltenyi Biotec; and CD45 (#11‐0451‐82) and CD206 (#12‐2061‐82), all were purchased from Thermo Fisher Scientific.

Techniques: Injection, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal:

Article Title: A defect in bone marrow derived dendritic cell maturation in the nonobesediabetic mouse

doi: 10.1046/j.1365-2249.2001.01473.x

Figure Lengend Snippet: Surface phenotype of bone marrow derived DC. The DC consisting of 90–95% CD11c+ cells were analysed by flow cytometry for the expression of I-A, β2-microglobulin, CD80, CD86 and CD40 (▪) or by staining with secondary antibody alone (□). Two I-A specific mAbs, Ox-6 (shown here) or 10·2.16 were used with identical result. The results are representative of three different experiments carried out three weeks apart.

Article Snippet: The antibodies used in the study were the MHC class II specific rat antimouse I-A k clone MRC Ox-6 (Serotec, Oxford, UK) and anti-I-A g7 clone 10·2.16 (kindly provided by EP Reich, Anergen Inc), rat antimouse CD80 (B7·1) clone IG10 (Pharmingen, San Diego, USA), rat antimouse CD86 (B7·2) clone GL1 (Cambridge Bioscience, UK); rat antimouse CD40 clone 3/23 (Serotec, UK), hamster antimouse CD11c clone N-418 (produced by ICRF, London, UK); FITC-conjugated rabbit antirat Ig (Dako, Ely, UK); FITC-conjugated rabbit anti‐mouse Ig (Dako, Denmark); and mouse anti‐mouse β2-microglobulin clone S19·8 (kindly provided by M Millrain, CSC, UK).

Techniques: Derivative Assay, Flow Cytometry, Expressing, Staining

Journal: bioRxiv

Article Title: Failed down-regulation of PI3K signaling makes autoreactive B cells receptive to bystander T cell help

doi: 10.1101/2023.01.23.525206

Figure Lengend Snippet: A) Schematic representation of the experimental protocol. One group of OTII x TCRα KO mice was treated with agonistic anti-CD40 antibody on days 6 and 9 post initiation of tamoxifen treatement. B) Proliferation and differentiation to plasmablasts of splenic YFP+ E4+ SHP-1 deficient B cells in WT C57BL/6 mice and OTII x TCRα KO mice, in the presence or absence of agonistic anti-CD40, 14 days after tamoxifen treatment (n=4-5/group, representative cytograms shown). C) Distribution of the YFP+ E4+ SHP-1 deficient Ars/A1 B cell population shown in between an undivided, proliferated and plasmablast (proliferated and CD138+) state. D) Quantification of antibody secreting cells (IgM a anti-Ars) by ELISPOT of spleen cells from . Gray area delineates the limit of detection (50 spots/spleen). E) Quantification of the number of CD45.1+ E4+ cells/spleen in the indicated transfers 14 days after tamoxifen treatment. F) Proliferation and differentiation to plasmablasts of splenic YFP+ E4+ WT Ars/A1 B cells in WT C57BL/6 mice, in the presence or absence of agonistic anti-CD40, 14 days after tamoxifen treatment (n=5/group, representative cytograms shown). G) Distribution of the YFP+ E4+ WT Ars/A1 B cell population shown in between an undivided, proliferated and plasmablast (proliferated and CD138+) state. H) Quantification of antibody secreting cells (IgM a anti-Ars) by ELISPOT of spleen cells from . Gray area delineates the limit of detection (50 spots/spleen). Data shown are representative of at least two replicate experiments. Error bars represent mean ± SEM. Two -tailed unpaired Student’s t test was used. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: In VivoMab anti-mouse CD40, (Clone:FGK4.5/FGK45; Bio X Cell) was injected i.v. or i.p. (200 μl at 0.25mg/ml in sterile PBS) for stimulation.

Techniques: Enzyme-linked Immunospot, Two Tailed Test

Journal: PLOS Pathogens

Article Title: Regulatory T cell-like response to SARS-CoV-2 in Jamaican fruit bats ( Artibeus jamaicensis ) transduced with human ACE2

doi: 10.1371/journal.ppat.1011728

Figure Lengend Snippet: Four bats were transduced with human ACE2 and infected 5 days later. After 12 days, splenocytes were recovered for AIM testing and gene expression analsysis. A. Splenocytes from hACE2-transduced bats 12 days post infection with SARS-CoV were cultured with or without SARS-CoV-2 nucleocapsid peptide library for 6 hours in the presence of anti-CD40 blocking antibody (AIM testing) or 24 hours without anti-CD40 blocking antibody (cellular RNA). B. Cells were stained with anti-CD154 and anti-CD4 and analyzed by flow cytometry. Violin plots for the individual bats, plus the pooled sample and rank data. In the presence of peptide, CD154 expression was significantly increased on splenocytes (red) compared to no peptide controls (blue) (Wilcoxon rank sum test). C. Heat map of splenocyte qPCR (ΔΔCq) gene expression in response to peptide stimulation determined 3 of the 4 bats expressed a profile consistent with a regulatory T cell response. D. Peptide-induced gene expression changes in splenocyte cultures of bats 1, 2 and 4 from panel C; IL-10, TGFβ and CD4 gene expression were significantly elevated, whereas CXCR4 was nearly significant (two-tailed t-test).

Article Snippet: Test samples were then resuspended in a 1:20 dilution of anti-mouse CD40 PE (Tonbo, clone FGK45), or anti-human CD154 PE (Tonbo, clone 5C8) in FACS buffer and incubated for 30 minutes at 4°C in the dark.

Techniques: Transduction, Infection, Gene Expression, Cell Culture, Blocking Assay, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: STAR Protocols

Article Title: CyTOF protocol for immune monitoring of solid tumors from mouse models

doi: 10.1016/j.xpro.2022.101949

Figure Lengend Snippet:

Article Snippet: Anti-Mouse CD40-161Dy dilution 1:50 , Standard BioTools , 3161020B.

Techniques: Blocking Assay, Purification, Recombinant, Staining, Sterility, Antibody Labeling, Software, Transferring, Microscopy