lymphoma b cell line ramos  (ATCC)

Journal: RSC Chemical Biology

Article Title: A novel SadP-scFv UCHT1 lectibody activates T cells and mediates lysis of Burkitt's lymphoma cells

doi: 10.1039/d5cb00027k

Figure Lengend Snippet: Binding and T cell-mediated target cell lysis induced by the SadP-scFv UCHT1 lectibody. (a) Representative flow cytometry histograms for target (Ramos, Namalwa) and effector (PBMCs) cells. The cells were incubated with increasing concentrations of SadP-scFv UCHT1 and stained with an anti-His-Tag-AF647 antibody to detect SadP-scFv UCHT1 at the cell surface. The SadP-scFv UCHT1 exhibited robust, dose-dependent binding to Gb3 + Ramos cells and PBMCs, while binding to Gb3 − Namalwa cells was abolished. (b) Ramos and (c) Namalwa cells were incubated together with PBMCs in a ratio of 5 : 1 (E : T) and SadP-scFv UCHT1 (0.2–10 nM) for 7 hours up to 48 hours. (b) At 24 hours post-incubation 15% of target cell lysis was recorded in presence of 1 nM of lectibody, reaching up to 42% for 10 nM. After 48 hours, the specific target cell lysis increased to 42% with 5 nM and 65% killing with 10 nM SadP-scFv UCHT1. The data are shown as the mean ± SEM ( N = 3) of four separate experiments. n = 4. (c) When the Gb3 − Namalwa cells were incubated with PBMCs in presence of the SadP-scFv UCHT1 no significant T cell-mediated target cell lysis was registered. (d) and (e) Comparison of the killing rates of Ramos cells triggered by the lectibody SadP-scFv UCHT1 (d) and StxB-scFv UCHT1 (e) in similar concentrations. The SadP lectibody achieved higher killing rates at lower concentrations and comparable cytotoxicity values to the StxB lectibody at higher concentrations. The data are shown as the mean ± SEM ( N = 3) of three separate experiments. n = 3. The experiments were performed with PBMCs derived from 3 different donors ( N = 3). Each dot represents the averaged data from each individual donor. Statistical significance was evaluated using a one-way ANOVA. p -values < 0.5 were considered significant. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Article Snippet: In this study, Burkitt's lymphoma-derived cell lines Ramos and Namalwa were used (Ramos cells, ACC 603, and Namalwa: CSN/70, ACC 70, DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Germany).

Techniques: Binding Assay, Lysis, Flow Cytometry, Incubation, Staining, Comparison, Derivative Assay

Journal: RSC Chemical Biology

Article Title: A novel SadP-scFv UCHT1 lectibody activates T cells and mediates lysis of Burkitt's lymphoma cells

doi: 10.1039/d5cb00027k

Figure Lengend Snippet: GCS inhibition by PPMP led to abolished target cell lysis. Namalwa cells still exhibit a residual, low Gb3 abundance therefore, Ramos cells were treated with a glucosylceramide synthesis (GCS) inhibitor called PPMP to abolish Gb3 completely. Ramos cells were treated with 2 µM PPMP for 72 hours before starting the incubation with PBMCs and the SadP-scFv UCHT1 treatment. (a), (b) To ensure that the Gb3 was no longer present at the cell surface, the cells were stained with (a) 2.6 nM StxB-AF647 and (b) 20 nM SadP which was detected using an anti-His-Tag-AF647 antibody. Cells receiving no PPMP and remaining unstained were used as a negative control (Ctrl neg). Cells not treated with PPMP (UTD) were stained with either StxB or SadP and used as a positive control. The staining with StxB showed no presence of Gb3 at the cell surface, whereas SadP exhibited a low amount of binding to PPMP-treated Ramos cells. (c) The PPMP treated Ramos cells were incubated as described above with PBMCs and SadP-scFv UCHT1 for 48 hours. No significant killing of target cells could be observed. The data are shown as the mean ± SEM ( N = 3) of three separate experiments. n = 3. Statistical significance was evaluated using a one-way ANOVA. p -values < 0.5 were considered significant. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Article Snippet: In this study, Burkitt's lymphoma-derived cell lines Ramos and Namalwa were used (Ramos cells, ACC 603, and Namalwa: CSN/70, ACC 70, DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Germany).

Techniques: Inhibition, Lysis, Incubation, Staining, Negative Control, Positive Control, Binding Assay

Journal: RSC Chemical Biology

Article Title: A novel SadP-scFv UCHT1 lectibody activates T cells and mediates lysis of Burkitt's lymphoma cells

doi: 10.1039/d5cb00027k

Figure Lengend Snippet: Upregulation of T cell activation markers CD69 (early) and CD71 (middle/late) in presence of the SadP-scFv UCHT1 lectibody. The T cells were incubated with the indicated target cell line and the lectibody. (a) At 18 hours post-incubation, cells were stained with anti-CD3-AF488 (1:200) and anti-CD69-APC (1:200) and analysed using flow cytometry. The MFI of CD3 + CD69 + cells was determined, and the antibody background was subtracted. The Fold increase was calculated from these values and compared to the control (PBMCs together with target cells, without lectibody). Incubation of T cells with Ramos cells and 10 nM lectibody for 18 hours lead to a significant increase in CD69 surface expression of about 6-fold compared to the control. Namalwa cells, on the other hand, did not elicit an increase of CD69 expression on the surface of the T cells in presence of the lectibody. (b) Incubation of PBMCs with SadP-scFV UCHT1 and Ramos cells for 24 hours led to an increase in CD69 fluorescence signal of 5-fold and 8-fold, respectively for both concentrations tested. There was no increase in CD69 signal when incubated with Namalwa cells. (c) After incubating T cells with SadP-scFv UCHT1 and Ramos cells for 24 hours the cells were stained with anti-CD3-AF647 (1:200) and anti-CD71-AF488 (1:200) and analysed by flow cytometry. The CD71 signal was increase significantly ∼7-fold using 5 nM and ∼10-fold using 10 nM SadP-scFv UCHT1. Incubation with Namalwa cells, again did not lead to a significant increase in CD71 signal after 24 hours. (d) Extending the incubation time in presence of Ramos cells to 48 hours resulted in an even greater fold increase (12-fold for 5 nM and 25-fold for 10 nM) while an incubation for 48 hours did not increase the CD71 signal in presence of Namalwa cells. Statistical significance was evaluated using a one-way ANOVA The data are shown as the mean ± SEM ( N = 3) of three separate experiments. n = 3. p -Values < 0.5 were considered significant. *** p ≤ 0.001; **** p ≤ 0.0001.

Article Snippet: In this study, Burkitt's lymphoma-derived cell lines Ramos and Namalwa were used (Ramos cells, ACC 603, and Namalwa: CSN/70, ACC 70, DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Germany).

Techniques: Activation Assay, Incubation, Staining, Flow Cytometry, Control, Expressing, Fluorescence