Journal: EMBO Molecular Medicine

Article Title: The MICA-129 dimorphism affects NKG2D signaling and outcome of hematopoietic stem cell transplantation

doi: 10.15252/emmm.201505246

Figure Lengend Snippet: MACS-separated CD8 + T cells were cultured in triplicate on an immobilized anti-CD3 mAb (0.005 μg/ml [upper panel] or 0.01 μg/ml [lower panel]) in combination with recombinant MICA-129Met-Fc, MICA-129Val-Fc, and OVA-Fc proteins at various concentrations (1.0, 0.5, 0.1, 0.0 μg/ml). After 72 h, 25% of the supernatant was harvested and IL-2 concentrations were measured by ELISA. The harvested medium was replaced by the same volume containing 1 μCi 3 H-labeled thymidine. After 12 h, the plates were completely harvested and the DNA-bound radioactivity was determined. The means and SD of the stimulation index (SI) are displayed ( n = 4). Significant differences between MICA-129Met/Val-Fc and OVA-Fc proteins were found when the antigen-specific signal (anti-CD3) was limited (* P < 0.05, t -test; upper left panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0372 and MICA-129Val-Fc versus OVA-Fc P = 0.0366; upper right panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0499 and MICA-129Val-Fc versus OVA-Fc P = 0.0192; 0.5 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0164 and MICA-129Val-Fc versus OVA-Fc P = 0.0357; lower left panel: 0.5 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0287 and MICA-129Val-Fc versus OVA-Fc P = 0.0232; lower right panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0171 and MICA-129Val-Fc versus OVA-Fc P = 0.0484). Purified CFSE-stained CD8 + T cells were stimulated by immobilized anti-CD3 (0.005 μg/ml) in combination with recombinant MICA-129Met-Fc, MICA-129Val-Fc, OVA-Fc proteins, or co-stimulatory mAb (anti-CD28, anti-NKG2D) or an isotype control (mIgG 1 ). The proliferation of CD3 + CD8 + T cells was assessed at 60 h by flow cytometry. Results of a representative out of 6 experiments are displayed. Untreated CFSE-stained CD8 + T cells are included for comparison. The percentage of proliferating cells and MFI for CFSE are indicated. The MFI of CFSE in unstimulated CD8 + T cells (control) was set to 100% in individual experiments ( n = 6), and the relative decrease due to proliferation was calculated. Means + SD are shown. Significant differences (* P = 0.0277, Wilcoxon test) between MICA-129Met-Fc versus MICA-129Val-Fc and OVA-Fc proteins were found at slightly higher anti-CD3 concentrations (0.1 and 0.05 μg/ml) than at later time points (see A). Anti-CD28 and anti-NKG2D mAb were used in parallel as a positive control, mean + SD are shown, and significant differences (* P = 0.0277, Wilcoxon test) to the isotype control (mIgG 1 ) are indicated ( n = 6).

Article Snippet: OVA-Fc (having a mIgG 2a Fc fragment), mIgG 2a (A111-3, BD Biosciences), and mIgG 1 (C76-47, BD Biosciences) served as controls.

Techniques: Cell Culture, Recombinant, Enzyme-linked Immunosorbent Assay, Labeling, Radioactivity, Purification, Staining, Flow Cytometry, Positive Control

Journal: EMBO Molecular Medicine

Article Title: The MICA-129 dimorphism affects NKG2D signaling and outcome of hematopoietic stem cell transplantation

doi: 10.15252/emmm.201505246

Figure Lengend Snippet: NKG2D expression on purified CD8 + T cells exposed to L-MICA-129Met ( n = 19) or L-MICA-129Val clones ( n = 19) for 0, 4, and 24 h was analyzed by flow cytometry. CD8 + T cells (2.5 × 10 5 ) were co-cultured with 5 × 10 4 target cells and analyzed for NKG2D expression after gating on CD3 + CD8 + T cells. The means and SD of the MFI of NKG2D (left panel) and of the percentage of NKG2D + CD8 + T cells (right panel) are displayed. Differences between the groups were analyzed by repeated measures ANOVA, and the P -values are indicated. Purified CD8 + T cells were cultured on plate-bound anti-NKG2D (1 μg/ml) or isotype control (mIgG 1 ) for 24 h before the NKG2D expression was measured by flow cytometry. Means and SD of MFI (upper panel) and percentage of NKG2D + cells (lower panel) are shown ( n = 6). Differences between the groups were analyzed by t -tests, and the P -values are indicated. These CD8 + T cells were subsequently CFSE-stained and cultured on plates coated with anti-CD3 (0.005 μg/ml) in combination with anti-CD28 (0.5 μg/ml) as a positive control or anti-NKG2D (0.5 μg/ml). Proliferation was measured after 60 h by flow cytometry. Untreated CFSE-stained CD8 + T cells are included for comparison. The percentage of proliferating cells and the MFI for CFSE are indicated. The MFI of CFSE in unstimulated CD8 + T cells (control) was set to 100% in individual experiments ( n = 6), and the relative decrease due to proliferation was calculated. Means + SD are shown. Significant differences (Wilcoxon test) between CD8 + T cells pre-exposed to anti-NKG2D and isotype control (mIgG 1 ) were found in these experiments at anti-CD3 concentrations of 0.01 and 0.005 μg/ml.

Article Snippet: OVA-Fc (having a mIgG 2a Fc fragment), mIgG 2a (A111-3, BD Biosciences), and mIgG 1 (C76-47, BD Biosciences) served as controls.

Techniques: Expressing, Purification, Clone Assay, Flow Cytometry, Cell Culture, Staining, Positive Control

Journal: Scientific Reports

Article Title: CEACAM1 mediates B cell aggregation in central nervous system autoimmunity

doi: 10.1038/srep29847

Figure Lengend Snippet: ( A,B ) CEACAM1 expression on B cells in the CNS in acute ( A ) and chronic ( B ) MP4-induced EAE. ( C ) CEACAM1 expression on B cells in lymph nodes of MP4-immunized mice. ( D ) Negative control staining in the absence of primary antibody. Scale bars: 50 μm. ( E ) Inhibition of murine B cell aggregation induced by 25 μg/ml LPS + 5 U/ml IL-4 after treatment with 200 μg/ml anti-CEACAM1 antibody (mCC1). For each experiment n = 3 B6 mice were pooled. B cell aggregation was induced over a period of 72 h (compare images of untreated and LPS + IL-4-stimulated B cells). Data are representative of four independent experiments with similar results. Statistical significance was determined by Wilcoxon rank sum test. Data are expressed as mean ± s.d. * P < 0.05; **P < 0.01; *** P < 0.001. ( F ) Clinical course of EAE after treatment of MP4-immunized B6 mice with mCC1 ( n = 8) or mIgG1 isotype control antibody ( n = 7). Treatment was started with disease onset and given every three days. Results are from three independent experiments. Statistical significance was determined by one-way ANOVA for day-to-day comparisons (* P < 0.05) or by Wilcoxon rank sum test for the entire time course ( ### P < 0.001). Data are expressed as mean ± s.d. ( G ) Representative image of perivascular infiltrates in the cerebellum of MP4-immunized mice (left panel, circles) and their number after treatment with mCC1 vs . mIgG (right panel). ( H–K ) Percentage of non-B cell infiltrates ( H ), diffuse B cell infiltrates ( I ) and B cell aggregates ( J ) and size of B cell aggregates ( K ) in the cerebellum of MP4-immunized mice after treatment with mCC1 vs . mIgG1. Statistical significance was determined by Wilcoxon rank sum test. * P < 0.05.

Article Snippet: MP4-immunized mice were treated by intraperitoneal injections of 150 μg anti-CEACAM1 mAb CC1 (mouse IgG1; produced and purified by inVivo BioTech Services) or isotype control mIgG1 (inVivo BioTech Services) every third day for 27.6 ± 5.4 days after disease onset.

Techniques: Expressing, Negative Control, Staining, Inhibition

Journal: Scientific Reports

Article Title: CEACAM1 mediates B cell aggregation in central nervous system autoimmunity

doi: 10.1038/srep29847

Figure Lengend Snippet: ( A ) B cell ELISPOT assay of draining inguinal lymph nodes for the detection of MP4-specific antibodies and total IgG in MP4-immunized mice treated with either mCC1 ( n = 8) or mIgG1 ( n = 7). ( B ) Serum ELISA for the detection of MP4-specific antibodies in MP4-immunized mice treated with either mCC1 ( n = 8) or mIgG1 ( n = 7). Statistical significance was determined by Wilcoxon rank sum test. ( C ) Representative images of IHC staining of inguinal draining lymph nodes from MP4-immunized mice treated either with mCC1 ( n = 8) or mIgG1 ( n = 7). Scale bars: 50 μm.

Article Snippet: MP4-immunized mice were treated by intraperitoneal injections of 150 μg anti-CEACAM1 mAb CC1 (mouse IgG1; produced and purified by inVivo BioTech Services) or isotype control mIgG1 (inVivo BioTech Services) every third day for 27.6 ± 5.4 days after disease onset.

Techniques: Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Journal: Science (New York, N.Y.)

Article Title: MAIT cells are imprinted by the microbiota in early life and promote tissue repair

doi: 10.1126/science.aax6624

Figure Lengend Snippet: (A-C) S. epidermidis LM061 (S. epi) was topically applied to the skin of WT mice on days 0, 2, 4, and 6. Fourteen days after the initial application, animals were compared to unassociated (control) mice. (A) Flow cytometry of TCRβ+ lymphocytes (top), IL-17A production by MAIT cells (middle), and coreceptor expression of MAIT cells (bottom) within the skin. (B) Number of cutaneous MAIT cells and (C) IL-17A+ MAIT cells. (D) Number of cutaneous MAIT cells following topical association with CD8+ T cell-inducing (LM087) and non-inducing (05001 and LM061) strains of S. epidermidis. (E) Both Lta−/− and WT mice were topically associated with S. epidermidis LM087 and the percentage change of T cells in the skin of Lta−/− mice compared to WT controls is depicted. Statistics denote whether the percentage differs significantly from the WT mean (100%). (F-G) (F) CD45.1 (5.1) and CD45.2 (5.2) mice were topically associated with S. epidermidis LM087, conjoined 7 weeks later, and analyzed 13 weeks following parabiosis. (G) Frequency of T cells in the indicated tissues of parabiotic mice that are host-derived. (H) WT mice were injected subcutaneously with 1 mg of either anti-IL-23R antibody or mIgG1 isotype control 2 days before the initial application of S. epidermidis LM061 on day 0 and again on day 6. Number of cutaneous MAIT cells is depicted. (I) Flow cytometry of TCRβ+ lymphocytes from the skin of WT mice. (J-L) WT mice were injected intraperitoneally with either 1 mg of anti-IL-18 antibody or saline (vehicle) 2 days before the initial application of S. epidermidis LM061 on day 0 and again on days 1, 5, 8, and 11. (J) Number of cutaneous MAIT cells and (K) IL-17A+ MAIT cells and (L) percentage of MAIT cells that are IL-17A+ in anti-IL-18-treated and control mice (vehicle) that were associated with S. epidermidis LM061. (M-O) (M) Number of cutaneous MAIT cells and (N) IL-17A+ MAIT cells in Il1r1−/− mice and WT controls associated with S. epidermidis LM061. (O) Flow cytometry of IL-17A production by MAIT cells within the skin of S. epidermidis-associated mice. (P-R) RNA-sequencing data of cutaneous MAIT cells from mice associated with S. epidermidis LM061 and unassociated controls. GO terms enriched in MAIT cells from S. epidermidis-associated mice that are related to (P) leukocyte activation and (Q) tissue repair. Positive (pos.), negative (neg.), and regulation (reg.) are abbreviated. (R) Heatmap depicting relative expression (exp.) of genes associated with tissue repair and leukocyte activation (Act.). Flow cytometry gate frequencies and graphs indicate means ± SEM. Data represent at least two experiments with four or more mice per group. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 as calculated by Student’s t-test. “ns” denotes that comparison was not significant.

Article Snippet: Mice were injected subcutaneously with 1 mg of either anti-IL-23R antibody (21A4; Merck) or mIgG1 isotype control (27F11; Merck) 2 d before the initial application of S. epidermidis NIHLM061 and again on day 6.

Techniques: Flow Cytometry, Expressing, Derivative Assay, Injection, RNA Sequencing Assay, Activation Assay

Journal: The Journal of Clinical Investigation

Article Title: Angiopoietin-2 blockade ameliorates autoimmune neuroinflammation by inhibiting leukocyte recruitment into the CNS

doi: 10.1172/JCI130308

Figure Lengend Snippet: ( A ) Ang2 protein concentration in the serum and SC lysates at different time points after EAE induction (0 dpi: n = 4; 7 dpi: n = 4; 14 dpi: n = 5; 21 dpi: n = 4; 28 dpi: n = 3). ( B ) Clinical scores and percentage of body weight loss of control (Ctrl, n = 9) versus EC-Ang2 ( n = 11) mice induced with active EAE. ( C ) Clinical scores and percentages of body weight loss of mice induced with active EAE and treated with mIgG1 versus Ang2 Ab prophylactically (starting at the time of EAE induction; 0 dpi) ( n = 10 per group). ( D ) Clinical scores of mice induced with active EAE and treated with mIgG1 versus Ang2 Ab preemptively (starting during the effector phase of EAE at 7 dpi) ( n = 10 per group). ( E and F ) Representative images and quantifications of MBP staining to show loss of myelin in the SC white matter from both prophylactic 14 dpi and preemptive 28 dpi groups ( n = 10 per group). Scale bars: 100 μm. ( G ) Clinical scores of mice induced with adoptive transfer EAE and treated with mIgG1 versus Ang2 Ab starting at the time of adoptive transfer. Data are pooled from 2 independent experiments ( n = 16 per group). Arrows indicate Ab injections. Mean ± SEM, 1-way ANOVA with Dunnett’s post hoc test for multiple comparisons ( A ), nonparametric Mann-Whitney U test ( B - D , and G , comparison of AUC values of clinical EAE scores over the disease course), 2-way repeated measures ANOVA ( B and C , body weight loss), and 2-tailed Student’s t test ( E and F ). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: mIgG1 isotype control Ab (Eli Lilly and Co.), mouse anti-mouse Ang2 Ab (18E5, Eli Lilly and Co.), hIgG1 isotype control Ab (Synagis, AbbVie), and ABTAA ( ) were administrated i.p. at a dose of 20–25 mg/kg body weight 2 to 3 times a week.

Techniques: Protein Concentration, Control, Staining, Adoptive Transfer Assay, MANN-WHITNEY, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Angiopoietin-2 blockade ameliorates autoimmune neuroinflammation by inhibiting leukocyte recruitment into the CNS

doi: 10.1172/JCI130308

Figure Lengend Snippet: ( A ) Flow cytometric quantification of the number of immune cells in the SCs of mIgG1- versus Ang2 Ab–treated EAE mice at 12 dpi ( n = 10 per group). ( B ) RT-qPCR quantification of mRNA levels of Th signature cytokines ( Ifng , Tnf , Il4 , and Il17a ) and integrin subunits ( Itga4 and Itgb1 ) in the SCs of mIgG1- versus Ang2 Ab–treated EAE mice at 14 dpi ( n = 10 per group). ( C ) Flow cytometric quantification of the number of immune cells in the SCs of control ( n = 6) versus EC-Ang2 ( n = 9) EAE mice at 12 dpi. ( D and E ) Representative immunofluorescent images and quantifications of Iba1 + microglia and macrophages, Ly-6G + granulocytes, and CD4 + Th cells in the SCs of mIgG1- versus Ang2 Ab–treated control and EAE mice ( n = 10 per group) at 14 dpi as well as in the SCs of control ( n = 7) versus EC-Ang2 ( n = 8) control and EAE mice at 12 dpi. Scale bars: 100 μm. Mean ± SEM, 2-tailed Student’s t test ( A - E ), and 2-way ANOVA with Bonferroni’s post hoc test for multiple comparisons (Iba1 staining in D and E ). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: mIgG1 isotype control Ab (Eli Lilly and Co.), mouse anti-mouse Ang2 Ab (18E5, Eli Lilly and Co.), hIgG1 isotype control Ab (Synagis, AbbVie), and ABTAA ( ) were administrated i.p. at a dose of 20–25 mg/kg body weight 2 to 3 times a week.

Techniques: Quantitative RT-PCR, Control, Staining

Journal: The Journal of Clinical Investigation

Article Title: Angiopoietin-2 blockade ameliorates autoimmune neuroinflammation by inhibiting leukocyte recruitment into the CNS

doi: 10.1172/JCI130308

Figure Lengend Snippet: ( A ) T-distributed stochastic neighbor embedding (t-SNE) analysis of main immune cell clusters in the SCs of mIgG1- versus Ang2 Ab– treated EAE mice at 14 dpi and single transgenic control versus EC-Ang2 EAE mice at 12 dpi. Heatmap showing log 2 expression of known marker genes for each cluster. ( B and C ) Relevant GO biological processes of significantly (adjusted P < 0.05) downregulated genes after Ang2 blockade and upregulated genes after Ang2 overexpression in microglia ( B ) and macrophages ( C ). ( D and E ) Venn diagram illustrating the number of genes that were regulated by Ang2 in microglia and macrophages. ( F ) Violin plots showing mRNA expression of significantly (adjusted P < 0.05) downregulated APOE-induced and MHCII-associated genes after Ang2 blockade. IgG, mIgG1; A2, Ang2 Ab. ( G ) Representative images and quantifications of MHCII immunostaining in Iba1 + cells in the SCs of mIgG1- versus Ang2 Ab–treated EAE mice at 14 dpi ( n = 10 per group) and control ( n = 7) versus EC-Ang2 ( n = 8) EAE mice at 12 dpi. Scale bars: 100 μm. ( H ) Representative flow cytometry overlay plots and quantifications showing GMFI of MHCII expression in microglia and macrophages in the SCs of mIgG1- versus Ang2 Ab–treated EAE mice at 14 dpi ( n = 10 per group) and control versus EC-Ang2 EAE mice at 12 dpi ( n = 8 per group). Mean ± SEM, 2-tailed Student’s t test ( G and H ). * P < 0.05; ** P < 0.01.

Article Snippet: mIgG1 isotype control Ab (Eli Lilly and Co.), mouse anti-mouse Ang2 Ab (18E5, Eli Lilly and Co.), hIgG1 isotype control Ab (Synagis, AbbVie), and ABTAA ( ) were administrated i.p. at a dose of 20–25 mg/kg body weight 2 to 3 times a week.

Techniques: Transgenic Assay, Control, Expressing, Marker, Over Expression, Immunostaining, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: Angiopoietin-2 blockade ameliorates autoimmune neuroinflammation by inhibiting leukocyte recruitment into the CNS

doi: 10.1172/JCI130308

Figure Lengend Snippet: ( A ) Violin plots showing expression of Angpt2 and its receptors Tek , Itga5 , and Itgb1 in EC, microglia, and macrophage clusters of mIgG1- and Ang2 Ab–treated EAE mice. ( B ) GMFI of Tie2 and α 5 integrin on the surface of ECs, macrophages, and microglia from naive versus EAE mice (naive, n = 4; EAE, n = 3) at 14 dpi as analyzed by flow cytometry. ( C ) Flow cytometric analysis of active cell surface integrin (FN7-10 binding) relative to total cell surface α 5 β 1 integrin in microglia and macrophages from mIgG1- and Ang2 Ab–treated EAE mice (mIgG1, n = 8; Ang2 Ab, n = 7) at the disease peak (16 dpi). Mean ± SEM, 2-way ANOVA with Bonferroni’s post hoc test for multiple comparisons ( B ) and 2-tailed Student’s t test ( C ). * P < 0.05; *** P < 0.001.

Article Snippet: mIgG1 isotype control Ab (Eli Lilly and Co.), mouse anti-mouse Ang2 Ab (18E5, Eli Lilly and Co.), hIgG1 isotype control Ab (Synagis, AbbVie), and ABTAA ( ) were administrated i.p. at a dose of 20–25 mg/kg body weight 2 to 3 times a week.

Techniques: Expressing, Flow Cytometry, Binding Assay

Journal: The Journal of Clinical Investigation

Article Title: Angiopoietin-2 blockade ameliorates autoimmune neuroinflammation by inhibiting leukocyte recruitment into the CNS

doi: 10.1172/JCI130308

Figure Lengend Snippet: ( A and B ) t-SNE analysis of the main EC clusters (venous and arterial) in the SCs of mIgG1- versus Ang2 Ab–treated EAE mice at 14 dpi and single transgenic control versus EC-Ang2 EAE mice at 12 dpi. capillary-V, capillary-venous; capillary-A, capillary-arterial. ( C ) Heatmap showing log 2 expression of known marker genes in each cluster. ( D and E ) Relevant GO biological processes of significantly (adjusted P < 0.05) downregulated genes after Ang2 blockade ( D ) and upregulated genes after Ang2 overexpression ( E ) in the SC capillary-venous ECs. ( F ) Venn diagram illustrating the number of genes that were regulated by Ang2 in the SC capillary-venous ECs. ( G ) Violin plots showing Vcam1 expression in different EC clusters as well as its differential expression in capillary-venous ECs regulated by Ang2. ( H and I ) Representative images (EAE) and quantification of VCAM1 in the SC blood vessels of mIgG1- versus Ang2 Ab–treated control ( n = 6 per group) and EAE ( n = 10 per group) mice at 14 dpi and control versus EC-Ang2 control (Ctrl, n = 3; EC-Ang2 , n = 4) and EAE (Ctrl, n = 7; EC-Ang2 , n = 8) mice at 12 dpi. Scale bars: 100 μm. Mean ± SEM, 2-way ANOVA with Bonferroni’s post hoc test for multiple comparisons ( H and I ). *** P < 0.001.

Article Snippet: mIgG1 isotype control Ab (Eli Lilly and Co.), mouse anti-mouse Ang2 Ab (18E5, Eli Lilly and Co.), hIgG1 isotype control Ab (Synagis, AbbVie), and ABTAA ( ) were administrated i.p. at a dose of 20–25 mg/kg body weight 2 to 3 times a week.

Techniques: Transgenic Assay, Control, Expressing, Marker, Over Expression, Quantitative Proteomics

Journal: The Journal of Clinical Investigation

Article Title: Angiopoietin-2 blockade ameliorates autoimmune neuroinflammation by inhibiting leukocyte recruitment into the CNS

doi: 10.1172/JCI130308

Figure Lengend Snippet: ( A and B ) Representative images and quantifications of Evans blue leakage in the SCs of naive ( n = 3), mIgG1- versus Ang2 Ab–treated EAE mice ( n = 5 per group) and control versus EC-Ang2 control ( n = 6 per group) and EAE ( n = 7 per group) mice at 12 dpi. ( C ) Representative images and quantification of extravascular TER-119 + RBCs in the SCs of control ( n = 6) versus EC-Ang2 ( n = 8) EAE mice at 12 dpi. Scale bars: 100 μm. Mean ± SEM, 1- or 2-way ANOVA with Bonferroni’s post hoc test for multiple comparisons ( A and B ) and 2-tailed Student’s t test ( C ). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: mIgG1 isotype control Ab (Eli Lilly and Co.), mouse anti-mouse Ang2 Ab (18E5, Eli Lilly and Co.), hIgG1 isotype control Ab (Synagis, AbbVie), and ABTAA ( ) were administrated i.p. at a dose of 20–25 mg/kg body weight 2 to 3 times a week.

Techniques: Control