Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The tumor antigen N -glycolyl-GM3 is a human CD1d ligand capable of mediating B cell and natural killer T cell interaction

doi: 10.1007/s00262-016-1812-y

Figure Lengend Snippet: CD1d interacts with NGcGM3. a NGcGM3 was adsorbed on 96-well microtiter plates that were incubated with either recombinant CD1d-IgG1 fusion or IgG1 isotype control. Detection of immobilized CD1d by NGcGM3-coated surfaces was performed with a biotinylated aIgG1, extravidin alkaline phosphatase, and p-nitrophenyl phosphate. Bars show the mean absorbance at 405 nm ± standard error of the mean (SEM) of 10 wells per treatment. For a control, we used wells that were not coated with NGcGM3 and were only coated with MeOH. **p < 0.01, t test. b Competitive ELISA assays with CD1d-IgG1 fusion that was pre-incubated ON with increasing amounts of NGcGM3 in solution (0, 5, 10, 50, 250 and 500 µg/mL) and subsequently added to the NGcGM3 adsorbed plates. The assays were performed in duplicate. c Competitive ELISA assays with 18:1 Biotinyl PE (2 µg/mL). Plates were coated with goat anti-mouse IgG1 Ab, and CD1d-IgG1 fusion was pre-incubated ON in mixtures containing control lipid (2 µg/mL) in the presence of NGcGM3 (competitor lipid) at the indicated concentrations; the mixtures were subsequently added to the anti-mouse IgG1 adsorbed plates. The assays were performed in duplicate. d Top panel flow cytometry analysis of NGcGM3 expression on the murine myeloma cell line X63. The cell line was stained with isotype-matched control mAb (gray-filled histogram) or 14F7 mAb (black line) followed by PE-conjugated aIgG1. The numbers represent the percentages of NGcGM3-expressing cells. Data are representative of three independent experiments. Lower panels flow cytometry analysis of recombinant CD1d-IgG1 fusion binding to the surface of the murine myeloma cell line X63. The cell line was incubated for 24 hs with isotype-matched control mAb (right panel, IgG1 black line) or CD1d-IgG1 (left panel, black line). Detection of CD1d bound to the X63 cells surface was evidenced by PE-conjugated aIgG1 staining (referred to as aCD1d in the figure). The staining controls were fresh cells stained with PE-conjugated aIgG1 (gray-filled histogram). Data are representative of three independent experiments

Article Snippet: Commercial NGcGM3 (Enzo Life Sciences, catalogue number ALX-302-019, purity ≥98 % according to the product data sheet) was adsorbed by solvent methanol (MeOH) evaporation on the surfaces of 96-well microtiter plates (PolySorp, Nunc) and then washed for removal of unbound ganglioside.

Techniques: Incubation, Recombinant, Competitive ELISA, Flow Cytometry, Expressing, Staining, Binding Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The tumor antigen N -glycolyl-GM3 is a human CD1d ligand capable of mediating B cell and natural killer T cell interaction

doi: 10.1007/s00262-016-1812-y

Figure Lengend Snippet: Cells overexpressing CD1d bind to NGcGM3. a Flow cytometry analysis of NGcGM3 binding to the surface of the C1RCD1d cell line. Cells were incubated for 24 hs with empty liposomes (left panel) or with NGcGM3-loaded liposomes (right panel) followed by staining with a mAb (14F7, referred as aNGcGM3 in the figure, black line) or isotype-matched control mAb (gray-filled histogram). Detection of 14F7 bound to C1RCD1d cell surface was evidenced by PE-conjugated aIgG1 staining. Data are representative of three independent experiments. b Flow cytometry analysis of NGcGM3 binding to the surface of the C1RCD1d and C1R cell lines. Staining as in a with an untransfected C1R cell line. c Effect of PFA treatment on NGcGM3 binding to C1RCD1d cells. Control (untreated, left panel) and PFA-treated cells (right panel) were incubated with NGcGM3-loaded liposomes, which was followed by staining, as in a, for either NGcGM3 binding (black line) or isotype-matched controls (gray-filled histogram). Data are representative of three independent experiments. d Effect of PFA treatment on CD1d expression evaluated by C1RCD1d. The black line indicates cells that were fixed with 4 % PFA for 20 min at 4 °C and washed with PBS with 10 % FCS. Finally, they were stained with PE-conjugated aCD1d (clone CD1d42). The gray-filled histogram indicates untreated cells. Data are representative of three independent experiments

Article Snippet: Commercial NGcGM3 (Enzo Life Sciences, catalogue number ALX-302-019, purity ≥98 % according to the product data sheet) was adsorbed by solvent methanol (MeOH) evaporation on the surfaces of 96-well microtiter plates (PolySorp, Nunc) and then washed for removal of unbound ganglioside.

Techniques: Flow Cytometry, Binding Assay, Incubation, Liposomes, Staining, Expressing

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The tumor antigen N -glycolyl-GM3 is a human CD1d ligand capable of mediating B cell and natural killer T cell interaction

doi: 10.1007/s00262-016-1812-y

Figure Lengend Snippet: Primary Bc bind NGcGM3 and binding can be inhibited by anti-CD1d. a Tonsillar (left) and peripheral blood (right) sorted Bc were incubated for 24 hs with empty liposomes (gray line histogram) or with NGcGM3-loaded liposomes (black line histogram), followed by staining with a mAb (14F7, referred as aNGcGM3 in the figure) or isotype-matched control mAb (dashed histogram). The numbers represent the percentages of NGcGM3-binding Bc. Data are representative of three independent experiments. b Upper panels tonsillar (left) and peripheral blood (right) sorted Bc were pretreated for 2 hs at 37 °C with aCD1d mAb (clone CD1d42) prior treatment as in a. The numbers represent the percentages of NGcGM3-binding Bc. Data are representative of three independent experiments. Lower panel: bars show the ratio between the percentage of cells binding NGcGM3 when CD1d is blocked and when it is not (mean of three independent experiments ± SEM)

Article Snippet: Commercial NGcGM3 (Enzo Life Sciences, catalogue number ALX-302-019, purity ≥98 % according to the product data sheet) was adsorbed by solvent methanol (MeOH) evaporation on the surfaces of 96-well microtiter plates (PolySorp, Nunc) and then washed for removal of unbound ganglioside.

Techniques: Binding Assay, Incubation, Liposomes, Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The tumor antigen N -glycolyl-GM3 is a human CD1d ligand capable of mediating B cell and natural killer T cell interaction

doi: 10.1007/s00262-016-1812-y

Figure Lengend Snippet: Strategy used to enrich iNKT population from PBMCs. a Upper panels on the left, representative flow cytometry dot plot showing CD3 and CD56 expression by freshly isolated PBMCs gated on the CD3 population. The numbers indicate the percentages of CD3+CD56+ (NKT, dashed square) and CD3+CD56− (T cells) in an average donor. Right flow cytometry dot plot showing the percentages of iNKT (CD3+CD56+Vα24-JαQ+TCR) within the CD3+CD56+ population (NKT). The numbers indicate the percentages of iNKT within the NKT population in fresh PBMCs from an average donor. The dashed arrow indicates gated cells as indicated on the top of the panel. Lower panels same as above using α-GalCer-expanded cells, as indicated on the right, instead of fresh cells. b IFNγ secretion measured by ELISA on the sorted CD19+ and CD3+ (95 %) culture supernatant (5 days). c CD1d-IgG1 was incubated ON with NGcGM3-loaded liposomes (right) or empty liposomes (left) and then used to stain human expanded iNKT. Detection of CD1d-IgG1 bound to the cell surface, as evidenced by PE-conjugated aIgG1 staining (referred to as aCD1d in the figure). Data are representative of four independent experiments

Article Snippet: Commercial NGcGM3 (Enzo Life Sciences, catalogue number ALX-302-019, purity ≥98 % according to the product data sheet) was adsorbed by solvent methanol (MeOH) evaporation on the surfaces of 96-well microtiter plates (PolySorp, Nunc) and then washed for removal of unbound ganglioside.

Techniques: Flow Cytometry, Expressing, Isolation, Enzyme-linked Immunosorbent Assay, Incubation, Liposomes, Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The tumor antigen N -glycolyl-GM3 is a human CD1d ligand capable of mediating B cell and natural killer T cell interaction

doi: 10.1007/s00262-016-1812-y

Figure Lengend Snippet: Addition of NGcGM3 to co-cultures of Bc and iNKT induces proliferation that can be inhibited by aCD1d. a Representative flow cytometry dot plots showing expression of CD3 and Ki-67 in co-cultures of Bc and expanded iNKT in the presence of empty liposomes (left, referred as vehicle in the figure), NGcGM3-loaded liposomes (middle) and NGcGM3-loaded liposomes plus blocking aCD1d mAb (right). Numbers indicate the percentages of cells in the quadrants. b Histogram presenting the proportions of CD3+Ki-67+ cells in co-cultures, as in a, normalized to those of the empty liposomes (referred as vehicle in the figure) supplemented cultures. c Histogram presenting the proportions of CD19+Ki-67+ cells in co-cultures, as in a, normalized to those of the empty liposome (referred as vehicle in the figure) supplemented cultures. Data in b and c show the mean of the pool ratios ± SEM from four independent experiments. *p < 0.05 and **p < 0.01, unpaired t test

Article Snippet: Commercial NGcGM3 (Enzo Life Sciences, catalogue number ALX-302-019, purity ≥98 % according to the product data sheet) was adsorbed by solvent methanol (MeOH) evaporation on the surfaces of 96-well microtiter plates (PolySorp, Nunc) and then washed for removal of unbound ganglioside.

Techniques: Flow Cytometry, Expressing, Liposomes, Blocking Assay

Journal: Human Vaccines & Immunotherapeutics

Article Title: Induction of leukocyte infiltration at metastatic site mediates the protective effect of NGcGM3-based vaccine

doi: 10.4161/hv.29161

Figure Lengend Snippet: Figure 1. Irrespective of the route of administration the anti-metastatic effect of the NGcGM3/VSSP vaccine (in a spontaneous lung metastases murine model) is similar. C57BL/6 mice were inoculated with 3LL-D122 cells (2 x 105/mouse) into the right hind footpad, and treated twice with the NGcGM3/VSSP vaccine (Vac), either subcutaneous (Vac-SC) or intraperitoneal (Vac-IP), 7 and 21 d after tumor inoculation. Control mice were injected with PBS. Vac treated animals had significantly fewer pulmonary metastases measured as lung weight, relative to control group. Differences were not observed between the 2 routes (P > 0.05). Column bars represent mean values and error bars correspond to standard errors. The P value was calculated with ANOVA and the Tukey multiple comparison tests (***P < 0.001).

Article Snippet: In vitro assay: BM-cDC or BM-pDC were cultured in 96-well culture plates at 37 °C in the presence of LPS (Sigma), NGcGM3/VSSP, CpG-A (InVivoGen), or medium (IMDM, Gibco).

Techniques: Injection, Comparison

Journal: Human Vaccines & Immunotherapeutics

Article Title: Induction of leukocyte infiltration at metastatic site mediates the protective effect of NGcGM3-based vaccine

doi: 10.4161/hv.29161

Figure Lengend Snippet: Figure 2. CD4+ T cells are involved in the NGcGM3/VSSP vaccine anti-metastatic effect. Depletion of T cells was achieved injecting anti CD4 mAb, 1 d after each vaccine immunization. Column bars represent mean values and error bars correspond to the standard error. The P value was calculated with ANOVA and the Tukey multiple comparison tests (*P < 0.05).

Article Snippet: In vitro assay: BM-cDC or BM-pDC were cultured in 96-well culture plates at 37 °C in the presence of LPS (Sigma), NGcGM3/VSSP, CpG-A (InVivoGen), or medium (IMDM, Gibco).

Techniques: Comparison

Journal: Human Vaccines & Immunotherapeutics

Article Title: Induction of leukocyte infiltration at metastatic site mediates the protective effect of NGcGM3-based vaccine

doi: 10.4161/hv.29161

Figure Lengend Snippet: Figure 3. The NGcGM3/VSSP vaccine promotes the increase, maturation, and cytokine secretion of Bone Marrow-derived conventional-DC in vitro and in vivo. BM cells of C57BL/6 mice were differentiated in GM-CSF-enriched medium. Cells were cultured overnight in the presence of LPS-1 µg/mL (C+), NGcGM3/VSSP (VAC-1 µg/mL), or medium (im-cDC). (A) Frequency of mature CD11c+ cells. (B) IL-6 and IL-12p40 secretion by NGcGM3/VSSP treated cDC in culture. Data are representative of 2 independent experiments. (C) C57BL/6 mice were injected sc with the NGcGM3/VSSP vaccine. The bar diagram shows the percentage of mature CD11c+ cells in draining lymph nodes, 3 d after Vac or PBS (control) treatments.

Article Snippet: In vitro assay: BM-cDC or BM-pDC were cultured in 96-well culture plates at 37 °C in the presence of LPS (Sigma), NGcGM3/VSSP, CpG-A (InVivoGen), or medium (IMDM, Gibco).

Techniques: Derivative Assay, In Vitro, In Vivo, Cell Culture, Injection

Journal: Human Vaccines & Immunotherapeutics

Article Title: Induction of leukocyte infiltration at metastatic site mediates the protective effect of NGcGM3-based vaccine

doi: 10.4161/hv.29161

Figure Lengend Snippet: Figure 4. The NGcGM3/VSSP vaccine increases the maturation of Bone Marrow-derived plasmacytoid Dendritic cells. BM of C57BL/6 mice were differentiated in FLT3L-enriched medium. B220+ purified cells were cultured overnight in the presence of LPS (0.5 µg/mL), CpG-A (5 µg/mL), NGcGM3/VSSP (Vac 0.1 µg/mL), or medium (im-pDC). (A) Frequency of CD86+B220+ cells. (B) IFNα secretion by NGcGM3/VSSP treated pDC in culture. Data are representative of 2 independent experiments. (C) C57BL/6 mice were injected ip with the NGcGM3/VSSP vaccine and serum levels of IFNα, IL6 and IL12p40 were determined by ELISA at 2-h intervals.

Article Snippet: In vitro assay: BM-cDC or BM-pDC were cultured in 96-well culture plates at 37 °C in the presence of LPS (Sigma), NGcGM3/VSSP, CpG-A (InVivoGen), or medium (IMDM, Gibco).

Techniques: Derivative Assay, Purification, Cell Culture, Injection, Enzyme-linked Immunosorbent Assay