Journal: World Journal of Surgical Oncology

Article Title: A novel sialyl Le X expression score as a potential prognostic tool in colorectal cancer

doi: 10.1186/1477-7819-10-95

Figure Lengend Snippet: Showcase the image sections of the five typical immunohistochemical staining patterns of sialyl Le X (100x magnification).

Article Snippet: As primary antibody, the monoclonal anti-sialyl Le X mouse-immunoglobulin M (IgM) (clone KM93, Kamiya Biomedical Company, Seattle, WA, USA) was used.

Techniques: Immunohistochemical staining, Staining

Journal: World Journal of Surgical Oncology

Article Title: A novel sialyl Le X expression score as a potential prognostic tool in colorectal cancer

doi: 10.1186/1477-7819-10-95

Figure Lengend Snippet: Clinical and histopathological criteria stratified by sialyl Le X expression

Article Snippet: As primary antibody, the monoclonal anti-sialyl Le X mouse-immunoglobulin M (IgM) (clone KM93, Kamiya Biomedical Company, Seattle, WA, USA) was used.

Techniques: Expressing

Journal: World Journal of Surgical Oncology

Article Title: A novel sialyl Le X expression score as a potential prognostic tool in colorectal cancer

doi: 10.1186/1477-7819-10-95

Figure Lengend Snippet: Cancer-related survival after resection of colorectal cancer depending on sialyl Le X expression stages III and IV (A), stage III alone (B) and stage IV alone (C).

Article Snippet: As primary antibody, the monoclonal anti-sialyl Le X mouse-immunoglobulin M (IgM) (clone KM93, Kamiya Biomedical Company, Seattle, WA, USA) was used.

Techniques: Expressing

Journal: eLife

Article Title: Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells

doi: 10.7554/eLife.11765

Figure Lengend Snippet: ( A ) A glycan profile of OVA was generated using a multidimensional normal phase nano-HPLC coupled with an electrospray ionization interface mass spectrometer with an intercalated nanofluorescence detector. The different glycan species, indicated by numbers, are shown on the right; their relative proportion is represented in a pie chart. ( B ) ELISA showing functional modification of OVA with Le X glycans, as detected with anti-Le X antibodies and resulting in binding of MGL1-Fc. Unconjugated OVA does not carry any ligands for MGL1. Modification of OVA with Le X did not alter the ability to bind to MR as illustrated by equal binding kinetics of MR-Fc to native OVA and OVA-Le X . OVA and OVA-Le X preparations contain similar amounts of OVA as detected with anti-OVA antibodies. DOI: http://dx.doi.org/10.7554/eLife.11765.003

Article Snippet: Unconjugated mouse anti-OVA (Sigma Aldrich), mouse anti-Le X (Calbiochem), rat anti-mMGL (ER-MP23; kind gift from Dr. P. Leenen, Erasmus MC, Rotterdam, The Netherlands), rat anti-LAMP1 (BD-Pharmingen), rabbit anti-Rab11 (Life Technologies), goat anti-EEA1 (Santa Cruz Biotechnology) and rabbit anti-EEA-1 (Dianova).

Techniques: Generated, Mass Spectrometry, Enzyme-linked Immunosorbent Assay, Functional Assay, Modification, Binding Assay

Journal: eLife

Article Title: Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells

doi: 10.7554/eLife.11765

Figure Lengend Snippet: ( A ) Expression of murine MGL on BM-DCs and CD11c + spDCs was analyzed by flow cytometry. ( B ) MGL1 mRNA expression by BM-DC and splenic DC from WT and MGL1 KO mice was determined using qRT-PCR. GAPDH was used as a reference gene and the results are representative of three independent experiments. C57BL/6 mice were immunized s.c. with either OVA-Le X or native OVA mixed with anti-CD40 using a prime-boost protocol. Spleens were analyzed by flow cytometry to determine the frequency of ( C ) H2-K b /SIINFEKL-tetramer-binding CD8 + T cells and IFN-γ or TNF production by activated CD8 + T cells was determined by intracellular staining after OVA-specific re-stimulation ex vivo. Dots represent individual mice (n=4–5 mice/group; **p<0.01). Bars indicate median of each group. Graphs shown are representative of two independent experiments. ( D ) C57BL/6 and MGL1 KO mice were prime-boosted with either OVA-Le X or native OVA mixed with anti-CD40. Frequencies of IFN-γ and TNF-double-producing CD8 + T cells were determined by intracellular staining after OVA-specific re-stimulation of splenocytes ex vivo. Dots represent individual mice (n=4–5 mice/group; *p<0.05 ***p<0.001). Bars indicate median of each group. Data are representative of 2 independent experiments. DOI: http://dx.doi.org/10.7554/eLife.11765.005

Article Snippet: Unconjugated mouse anti-OVA (Sigma Aldrich), mouse anti-Le X (Calbiochem), rat anti-mMGL (ER-MP23; kind gift from Dr. P. Leenen, Erasmus MC, Rotterdam, The Netherlands), rat anti-LAMP1 (BD-Pharmingen), rabbit anti-Rab11 (Life Technologies), goat anti-EEA1 (Santa Cruz Biotechnology) and rabbit anti-EEA-1 (Dianova).

Techniques: Expressing, Flow Cytometry, Quantitative RT-PCR, Binding Assay, Staining, Ex Vivo

Journal: eLife

Article Title: Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells

doi: 10.7554/eLife.11765

Figure Lengend Snippet: ( A ) Pulsing of CD11c + spDCs or BM-DCs with OVA-Le X results in equal OT-II proliferation as native OVA. Expansion of OVA-specific T cells was determined using 3 H-thymidine incorporation. Data are shown as mean ± SD of triplicate cultures, representative of three independent experiments. ( B ) Flow cytometric analysis of OT-II or DO11.10 T cells differentiated by OVA-Le X or OVA-loaded spDCs or BM-DCs. Cells were gated on CD4 + T cells. Numbers in dot plots indicate the percentage of IFN-γ + or IL-4 + of CD4 + T cells. Dot plots are representative of five independent experiments. ( C ) C57BL/6 mice were immunized s.c. with either OVA-Le X or native OVA mixed with anti-CD40 using a prime-boost protocol and the frequency of IFN-γ-producing activated CD4 + T cells in spleen was determined by intracellular staining after OVA-specific re-stimulation ex vivo. Dots represent individual mice, bars indicate median of each group (n=5 mice/group, **p<0.01). Graphs shown are representative of two independent experiments. DOI: http://dx.doi.org/10.7554/eLife.11765.008

Article Snippet: Unconjugated mouse anti-OVA (Sigma Aldrich), mouse anti-Le X (Calbiochem), rat anti-mMGL (ER-MP23; kind gift from Dr. P. Leenen, Erasmus MC, Rotterdam, The Netherlands), rat anti-LAMP1 (BD-Pharmingen), rabbit anti-Rab11 (Life Technologies), goat anti-EEA1 (Santa Cruz Biotechnology) and rabbit anti-EEA-1 (Dianova).

Techniques: Staining, Ex Vivo

Journal: eLife

Article Title: Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells

doi: 10.7554/eLife.11765

Figure Lengend Snippet: WT BM-DC were loaded with OVA-Le X or native OVA for 4h and subsequently co-cultured with OT-II T cells for six days. Proliferation of OT-II T cells was determined by [ 3 H]-thymidine uptake and presented as mean ± SD of triplicate cultures. Data shown are representative of two independent experiments. DOI: http://dx.doi.org/10.7554/eLife.11765.009

Article Snippet: Unconjugated mouse anti-OVA (Sigma Aldrich), mouse anti-Le X (Calbiochem), rat anti-mMGL (ER-MP23; kind gift from Dr. P. Leenen, Erasmus MC, Rotterdam, The Netherlands), rat anti-LAMP1 (BD-Pharmingen), rabbit anti-Rab11 (Life Technologies), goat anti-EEA1 (Santa Cruz Biotechnology) and rabbit anti-EEA-1 (Dianova).

Techniques: Cell Culture

Journal: eLife

Article Title: Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells

doi: 10.7554/eLife.11765

Figure Lengend Snippet: Control cells were incubated with medium. The percentage of gated antigen-positive DCs are indicated. Lower panel: histograms indicating the mean uptake of OVA (left, black line) or OVA-Le X (right, black line) versus medium (grey filled histograms) and EGTA (dashed lines) controls. Numbers indicate the MFI of OVA-positive DCs. DOI: http://dx.doi.org/10.7554/eLife.11765.012

Article Snippet: Unconjugated mouse anti-OVA (Sigma Aldrich), mouse anti-Le X (Calbiochem), rat anti-mMGL (ER-MP23; kind gift from Dr. P. Leenen, Erasmus MC, Rotterdam, The Netherlands), rat anti-LAMP1 (BD-Pharmingen), rabbit anti-Rab11 (Life Technologies), goat anti-EEA1 (Santa Cruz Biotechnology) and rabbit anti-EEA-1 (Dianova).

Techniques: Incubation

Journal: eLife

Article Title: Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells

doi: 10.7554/eLife.11765

Figure Lengend Snippet: BM-DCs and CD11c + spDCs loaded with OVA-Le X enhanced OT-I proliferation compared to native OVA loaded DCs. Proliferation was determined on day 3 by [ 3 H]-Thymidine uptake and presented as mean ± SD of triplicate cultures. Data are representative of four independent experiments. DOI: http://dx.doi.org/10.7554/eLife.11765.013

Article Snippet: Unconjugated mouse anti-OVA (Sigma Aldrich), mouse anti-Le X (Calbiochem), rat anti-mMGL (ER-MP23; kind gift from Dr. P. Leenen, Erasmus MC, Rotterdam, The Netherlands), rat anti-LAMP1 (BD-Pharmingen), rabbit anti-Rab11 (Life Technologies), goat anti-EEA1 (Santa Cruz Biotechnology) and rabbit anti-EEA-1 (Dianova).

Techniques:

Journal: eLife

Article Title: Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells

doi: 10.7554/eLife.11765

Figure Lengend Snippet: ( A ) Uptake of fluorescent-labeled OVA-Le X or native OVA (30 µg/ml) by WT BM-DCs was analyzed by flow cytometry after 90 min. Graphs indicate the mean ± SD of triplicates and are representative of three independent experiments. ( B ) CFSE-labeled OT-I T cells were incubated with BM-DCs pulse-loaded with indicated concentrations of OVA-Le X or OVA for 4h. Un-loaded DC served as controls. Proliferation of OT-I T cells was analyzed after 3 days by flow cytometry. Percentages of divided OT-I cells are indicated. ( C ) OVA-Le X induces more OVA257-264/H2-K b I complexes at the cell-surface of DCs than native OVA, as shown by 25.1D1 staining 18h after pulse loading of BM-DCs with OVA-Le X or native OVA. *p<0.05. ( D ) WT or MGL1 KO BM-DCs or CD11c + spDCs are pulsed with OVA-Le X (black bars) or native OVA and OT-I proliferation was determined on day 3 by [ 3 H]-thymidine uptake. Data are presented as mean ± SD of triplicates, representative of three independent experiments. ***p<0.001, ns not significant. ( E ) Cross-presentation of OVA-Le X is independent of MyD88 and/or TRIF signaling. BM-DC from WT or MyD88/TRIF DKO mice were pulsed with indicated concentrations of antigen and co-cultured with OT-I T cells. DCs pulsed with the nominal epitope SIINFEKL served as controls (right panel). Proliferation was determined by [ 3 H]-thymidine uptake. Data are representative of two experiments and indicated as mean ± SD of triplicates. DOI: http://dx.doi.org/10.7554/eLife.11765.011

Article Snippet: Unconjugated mouse anti-OVA (Sigma Aldrich), mouse anti-Le X (Calbiochem), rat anti-mMGL (ER-MP23; kind gift from Dr. P. Leenen, Erasmus MC, Rotterdam, The Netherlands), rat anti-LAMP1 (BD-Pharmingen), rabbit anti-Rab11 (Life Technologies), goat anti-EEA1 (Santa Cruz Biotechnology) and rabbit anti-EEA-1 (Dianova).

Techniques: Labeling, Flow Cytometry, Incubation, Staining, Cell Culture

Journal: eLife

Article Title: Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells

doi: 10.7554/eLife.11765

Figure Lengend Snippet: Both OVA and OVA-Le X were tested for endotoxin levels. Human embryonic kidney (HEK)293-TLR4/MyD88 transfectants (kind gift of Dr. D. Golenbock) were cultured in the presence of either antigen preparation (30 µg/ml) or indicated amounts of E. coli -derived LPS (Sigma Aldrich). The HEK transfectants respond to LPS by secreting IL-8. In both preparations, LPS was below detection limits (dashed line). Results are representative of two independent experiments. DOI: http://dx.doi.org/10.7554/eLife.11765.016

Article Snippet: Unconjugated mouse anti-OVA (Sigma Aldrich), mouse anti-Le X (Calbiochem), rat anti-mMGL (ER-MP23; kind gift from Dr. P. Leenen, Erasmus MC, Rotterdam, The Netherlands), rat anti-LAMP1 (BD-Pharmingen), rabbit anti-Rab11 (Life Technologies), goat anti-EEA1 (Santa Cruz Biotechnology) and rabbit anti-EEA-1 (Dianova).

Techniques: Cell Culture, Derivative Assay

Journal: eLife

Article Title: Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells

doi: 10.7554/eLife.11765

Figure Lengend Snippet: To examine whether cross-presentation of OVA-Le X involves TAP or Cathepsin-S ( A ) TAP1 KO and ( B ) Cat-S KO BM-DCs and WT BM-DCs were pulsed with OVA-Le X or native OVA and co-cultured with OT-I T cells for 3 days. DCs exogenously loaded with SIINFEKL for 3h served as control. Proliferation was determined by [ 3 H]-Thymidine uptake and data are presented as mean ± SD of triplicates (representative of three experiments). DOI: http://dx.doi.org/10.7554/eLife.11765.017

Article Snippet: Unconjugated mouse anti-OVA (Sigma Aldrich), mouse anti-Le X (Calbiochem), rat anti-mMGL (ER-MP23; kind gift from Dr. P. Leenen, Erasmus MC, Rotterdam, The Netherlands), rat anti-LAMP1 (BD-Pharmingen), rabbit anti-Rab11 (Life Technologies), goat anti-EEA1 (Santa Cruz Biotechnology) and rabbit anti-EEA-1 (Dianova).

Techniques: Cell Culture

Journal: eLife

Article Title: Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells

doi: 10.7554/eLife.11765

Figure Lengend Snippet: ( A, B ) WT BM-DCs were pulsed with AlexaFluor 674-OVA-Le X (30 µg/ml) and chased at the indicated time-points to assess triple co-localization scores of OVA-Le X with ( A ) EEA-1 and Rab11 or ( B ) LAMP1 and Rab11 using imaging flow cytometry. ( C ) WT (upper panels) and MGL1 KO (lower panels) BM-DCs were incubated with Dylight-633-OVA-Le X or native OVA (30 µg/ml) and 2h later co-localization of OVA antigen (Red) with early endosomal (EEA-1, Green) or endosomal/lysosomal (LAMP1, Green) and recycling endosomal (Rab11, Blue) compartments was analyzed using CSLM. From a z-stack, histograms were created for a selected area (indicated by a line, upper part of each panel) using the Leica confocal software. Histograms were created from each fluorochrome and overlays were made by the program. Arrows indicate co-localization of antigen (Red) with EEA1&Rab11 or LAMP1&Rab11. ( D ) MGL1-Fc binding to Le X -PAA was determined at indicated pH by ELISA. DOI: http://dx.doi.org/10.7554/eLife.11765.018

Article Snippet: Unconjugated mouse anti-OVA (Sigma Aldrich), mouse anti-Le X (Calbiochem), rat anti-mMGL (ER-MP23; kind gift from Dr. P. Leenen, Erasmus MC, Rotterdam, The Netherlands), rat anti-LAMP1 (BD-Pharmingen), rabbit anti-Rab11 (Life Technologies), goat anti-EEA1 (Santa Cruz Biotechnology) and rabbit anti-EEA-1 (Dianova).

Techniques: Imaging, Flow Cytometry, Incubation, Software, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells

doi: 10.7554/eLife.11765

Figure Lengend Snippet: WT BM-DCs were pulsed for 4 h with titrated amounts of OVA-Le X and washed with culture medium. DCs were then chased for 48 h in antigen-free medium. ( A ) BM-DCs pulsed for 4h with OVA-Le X induced MHC-I antigen presentation as measured by CFSE-labeled OVA-specific OT-I cells (upper panel). Sustained presentation is shown after 48 h (lower panel). ( B ) MHC-II antigen presentation 4h and 48h after pulse-loading with OVA-Le X , analyzed by OT-II proliferation. Data are presented as percentage of proliferated T cells and representative of three independent experiments. DOI: http://dx.doi.org/10.7554/eLife.11765.022

Article Snippet: Unconjugated mouse anti-OVA (Sigma Aldrich), mouse anti-Le X (Calbiochem), rat anti-mMGL (ER-MP23; kind gift from Dr. P. Leenen, Erasmus MC, Rotterdam, The Netherlands), rat anti-LAMP1 (BD-Pharmingen), rabbit anti-Rab11 (Life Technologies), goat anti-EEA1 (Santa Cruz Biotechnology) and rabbit anti-EEA-1 (Dianova).

Techniques: Labeling