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: Theranostics

Article Title: Adoptive therapy with amyloid-β specific regulatory T cells alleviates Alzheimer's disease

doi: 10.7150/thno.75965

Figure Lengend Snippet: Aβ-specific Tregs suppress Aβ-induced microglial inflammation via the bystander effect. (A) Representative images show the primary microglial activity after 30 min of Aβ (5 μM) treatment in the presence of CDr20. The scale bar represents 100 μm. (B) Fluorescence intensity of fifty randomly selected cocultured microglia and Treg cells after 30 min of Aβ treatment with CDr20 in the red fluorescent channel (excitation: 570 nm, emission: 600 nm). (C) The secreted levels of TNF-α and IL-1β in the supernatants of cocultured cells treated with Aβ for 6 h were measured by ELISA. (D) The relative mRNA expression levels of iNOS and CD86 in cocultured microglia and Treg cells were quantified. (E) Schematic diagram for the experiment using indirect coculture with a Transwell insert (0.4 μm) to confirm the inhibitory effect of Aβ + Tregs. The relative mRNA expression levels of (F) the proinflammatory cytokines IL-1β and TNF-α and (G) the microglial activation markers CD86 and iNOS were quantified in activated microglia after 6 hrs of coculture. (H) Schematic diagram for the Aβ + Treg migration assay. (I) The bar graph shows the percentage of Tregs that migrated toward Aβ compared to KLH. (J) Schematic diagram of the migration assay performed with Aβ + Tregs and anti-CD40 or anti-CD80 antibody-treated primary microglia. (K) The bar graph shows the percentage of Tregs that migrated toward antibody-treated microglia. Data are presented as the mean ± SEM. n = 3-4 per group; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: To block the interaction between Aβ-activated microglia and Tregs, we used the following antibodies as blocking antibodies: purified rat IgG (clone RTK2071; BioLegend, #400402), purified hamster anti-mouse CD40 (clone HM40-3; BD Biosciences, #553721), and purified rat anti-CD80 (clone 1G10/B7; BD Biosciences, #553368).

Techniques: Activity Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Expressing, Activation Assay, Migration

Journal: Clinical Cancer Research

Article Title: A Novel Chemoimmunomodulating Property of Docetaxel: Suppression of Myeloid-Derived Suppressor Cells in Tumor Bearers

doi: 10.1158/1078-0432.ccr-10-0733

Figure Lengend Snippet: Fig. 4. A, MDSCs from docetaxel-treated mice express M1 markers. MDSCs were affinity column purified from three mice per group representing naive mice, docetaxel-treated naive mice, tumor bearers, and docetaxel-treated tumor bearers. They were immunostained for CCR7 and iNOS to represent M1 markers or MR to represent a specific M2 marker before flow cytometric analysis. A, histogram representation of MR, CCR7, and iNOS levels in naive mice, docetaxel-treated naive mice, tumor bearers, and docetaxel-treated tumor bearers. B, columns, mean fluorescence intensity units of CCR7, MR, and iNOS from three independent experiments; bars, SE. C, in vivo docetaxel-treated MDSCs have increased differentiation markers. MACS column– purified MDSCs from the above treatment groups were analyzed by flow cytometry for MHC class II, CD11c, CD40, CD80, and CD85 expression. Gray filled histograms are isotype controls, dotted lines represent the docetaxel-treated MDSCs, and black solid lines represent untreated MDSCs.

Article Snippet: Anti-Ly6G and anti-Ly6C ylated antibodies, FITC-conjugated anti-LY6C, APCgated anti-LY6C, PE-conjugated anti-Ly6G, FITCgated anti-mouse CD40, PE-conjugated anti-mouse , FITC-conjugated anti-mouse CD80, APC-conjugated ouse CD11c, and PE-conjugated anti–mouse I Ad purchased from BD Biosciences; rat anti-mouse MR (CD206) was from AbD Serotec; and streptavidin beads were purchased from Miltenyi Biotec.

Techniques: Affinity Column, Purification, Marker, Fluorescence, In Vivo, Flow Cytometry, Expressing

Journal: Clinical and Vaccine Immunology

Article Title: Evidence of a Functional B-Cell Immunodeficiency in Adults Who Experience Serogroup C Meningococcal Disease

doi: 10.1128/cvi.00485-08

Figure Lengend Snippet: FIG. 3. T-cell help to B cells. B-cell activation in response to T-cell help was assessed by CD86 expression on cells stimulated with --dex along with T cells activated through CD3 and CD28. PBLs were incubated in the presence of various stimuli for 4 days, with some having previously been labeled with CFSE. Cells were then stained for CD19 and CD86. Mean increases in CD86 expression are shown for control or patient B cells after incubation of lymphocytes with either anti-CD3/CD28 (a) or both anti-CD3/CD28 and --dex (b). There was no significant difference in the ability of patient or control B cells to be activated in response to either CD3/CD28 stimulation alone or in response to the combination of --dex with the two T-cell stimuli. (c) PBLs from two donors were stimulated as described above, but with the addition of a blocking anti-CD154 monoclonal antibody or an isotype control antibody (mouse immunoglobulin) in some of the wells. (d and e) CD19 B-cell proliferation of patient or control B cells after incubation of PBLs with CD3/CD28-activated T cells (d) or with CD3/CD28 and --dex (b). There was no significant difference in the ability of patient or control B cells to proliferate in response to the T-cell activation signals alone, but additional proliferation in response to --dex was reduced in the patient cells in comparison with that in controls (P 0.015; Wilcoxon signed rank test).

Article Snippet: Anti-human CD154 antibody was purchased from R&D Systems Inc. - -Dex was prepared by us as described previously (20, 25).

Techniques: Activation Assay, Expressing, Incubation, Labeling, Staining, Control, Blocking Assay, Comparison

Journal: Science Advances

Article Title: STAT3 haploinsufficiency is associated with autosomal dominant hyper-IgE syndrome

doi: 10.1126/sciadv.adw2464

Figure Lengend Snippet: ( A ) STAT3 expression was evaluated by Western blot in Stat3 +/+ and Stat3 +/− cells derived from the spleen, thymus, and bone marrow (BM). Tubulin was used as loading control. Each lane represents one mouse. ( B ) A quantification of the STAT3/loading control ratio after normalization is shown for independently performed experiments as in (A). The mean ± SD is plotted. ( C ) IgE serum levels in Stat3 +/+ and Stat3 +/− mice were measured by ELISA. ( D ) Flow cytometry to identify IgG1 + and IgE + cells from IgM − IgD − cells 4 days after culturing Stat3 +/+ and Stat3 +/− B220 + splenocytes with IL-4 (12.5 ng/ml) and anti-CD40 antibody (62.5 ng/ml) in the absence or presence of IL-21 (25 ng/ml). Numbers in dot plots represent cell frequencies. ( E ) Mean (±SD) frequencies of the IgG1 + and IgE + cells as gated in (D). ( F ) Levels of IgG1 and IgE in the supernatant of cell cultures as done in (D) were measured by ELISA. B220 + splenocytes from Stat3 +/+ and Stat3 +/− mice were stimulated for different time points with IL-21 (50 ng/ml) ( G ), IL-10 (50 ng/ml) ( H ), and IL-6 (50 ng/ml) ( I ), and the phosphorylation of STAT3 (p-STAT3) was evaluated by flow cytometry. Mean (±SD) of mean fluorescent intensity (MFI) is plotted. Data are representative of at least three independent experiments. P values by unpaired t test (C) or two-way ANOVA followed by Bonferroni’s or Tukey’s multiple comparisons test (B and E to I).

Article Snippet: Cells were cultured in complete B cell medium [RPMI with 10% FBS, 10 mM Hepes, GlutaMAX, penicillin-streptomycin (Gibco), and 50 μM β-mercaptoethanol (Sigma-Aldrich), supplemented with IL-4 (12.5 ng/ml; PeproTech) and anti-CD40 antibody (62.5 ng/ml; Miltenyi Biotec), in the absence or presence of IL-21 (25 ng/ml; PeproTech)].

Techniques: Expressing, Western Blot, Derivative Assay, Control, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Phospho-proteomics