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Flow cytometric assessment of human B‐cell activation and differentiation. ( A) Representative density plots of B‐cell activation and differentiation on days 0, 4, 7, 10, and 14; human B cells from untreated control wells, Fab20‐containing wells, or Fab isotype control wells. The y ‐axis shows the CD95 signal. The x‐axis shows the CD38 signal. Three gates were created based on CD95 and CD38 status. CD95 − CD38 − cells (double negative) were labeled as “resting B cells”. CD95 + CD38 − B cells were labeled as “Activated B cells”, and CD95 + CD38 + (double positive) B cells were labeled as a subset resembling plasmablasts and plasma cells, “PB+PC”. <t>Naïve</t> <t>B</t> cells were CD95 − CD38 − (resting B cells). Density plots representative of three experiments ( n = 3). ( B) Representative density plots of IgD ( x ‐axis) and CD27 ( y ‐axis) status of B cells on days 0, 4, 7, 10, and 14. IgD + CD27 − B cells were classified as naïve B cells, and 97.1 % of B cells from baseline were IgD + CD27 − . Upon activation, B cells downregulate IgD and become IgD − CD27 − double negative B cells. CD27 (IgD − CD27 + ) was used as a marker for B‐cell differentiation into memory B cells and plasmablast subsets. Density plots representative of three experiments ( n = 3). ( C) CD95 MFI on B cells demonstrates how CD95 cannot be detected on the surface of naïve B cells (day 0) but is quickly upregulated upon stimulation. Fab20 completely inhibits CD95 upregulation. N = 3, bar graphs with whiskers equals mean ± SD. ( D) IgD MFI on B cells from the 14‐day assay. Day 0 B cells present with high IgD levels on the surface, but the expression is decreased to almost zero upon stimulation. Fab20 prevents IgD levels from decreasing, thus keeping B cells in a more naïve state. N = 3, bar graphs with whiskers equals mean ± SD. ( E) Immunoassay quantifying total IgG levels in supernatants from B cell cocultures on days 4, 7, 10, and 14 ( n = 4 from each timepoint, mean ± SD). On day 14, B‐cell differentiation caused untreated control and Fab isotype control B cells to produce high IgG levels. Fab20 completely prevented IgG production, also caused by the few surviving B cells on day 14. Unpaired t ‐tests of day 14 IgG levels, * p < 0.05, *** p < 0.01.
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Flow cytometric assessment of human B‐cell activation and differentiation. ( A) Representative density plots of B‐cell activation and differentiation on days 0, 4, 7, 10, and 14; human B cells from untreated control wells, Fab20‐containing wells, or Fab isotype control wells. The y ‐axis shows the CD95 signal. The x‐axis shows the CD38 signal. Three gates were created based on CD95 and CD38 status. CD95 − CD38 − cells (double negative) were labeled as “resting B cells”. CD95 + CD38 − B cells were labeled as “Activated B cells”, and CD95 + CD38 + (double positive) B cells were labeled as a subset resembling plasmablasts and plasma cells, “PB+PC”. Naïve B cells were CD95 − CD38 − (resting B cells). Density plots representative of three experiments ( n = 3). ( B) Representative density plots of IgD ( x ‐axis) and CD27 ( y ‐axis) status of B cells on days 0, 4, 7, 10, and 14. IgD + CD27 − B cells were classified as naïve B cells, and 97.1 % of B cells from baseline were IgD + CD27 − . Upon activation, B cells downregulate IgD and become IgD − CD27 − double negative B cells. CD27 (IgD − CD27 + ) was used as a marker for B‐cell differentiation into memory B cells and plasmablast subsets. Density plots representative of three experiments ( n = 3). ( C) CD95 MFI on B cells demonstrates how CD95 cannot be detected on the surface of naïve B cells (day 0) but is quickly upregulated upon stimulation. Fab20 completely inhibits CD95 upregulation. N = 3, bar graphs with whiskers equals mean ± SD. ( D) IgD MFI on B cells from the 14‐day assay. Day 0 B cells present with high IgD levels on the surface, but the expression is decreased to almost zero upon stimulation. Fab20 prevents IgD levels from decreasing, thus keeping B cells in a more naïve state. N = 3, bar graphs with whiskers equals mean ± SD. ( E) Immunoassay quantifying total IgG levels in supernatants from B cell cocultures on days 4, 7, 10, and 14 ( n = 4 from each timepoint, mean ± SD). On day 14, B‐cell differentiation caused untreated control and Fab isotype control B cells to produce high IgG levels. Fab20 completely prevented IgG production, also caused by the few surviving B cells on day 14. Unpaired t ‐tests of day 14 IgG levels, * p < 0.05, *** p < 0.01.

Journal: European Journal of Immunology

Article Title: Curbing Autoimmunity: A New Fab Fragment Targeting CD40‐CD40L Halts B‐Cell Activation and Differentiation

doi: 10.1002/eji.70158

Figure Lengend Snippet: Flow cytometric assessment of human B‐cell activation and differentiation. ( A) Representative density plots of B‐cell activation and differentiation on days 0, 4, 7, 10, and 14; human B cells from untreated control wells, Fab20‐containing wells, or Fab isotype control wells. The y ‐axis shows the CD95 signal. The x‐axis shows the CD38 signal. Three gates were created based on CD95 and CD38 status. CD95 − CD38 − cells (double negative) were labeled as “resting B cells”. CD95 + CD38 − B cells were labeled as “Activated B cells”, and CD95 + CD38 + (double positive) B cells were labeled as a subset resembling plasmablasts and plasma cells, “PB+PC”. Naïve B cells were CD95 − CD38 − (resting B cells). Density plots representative of three experiments ( n = 3). ( B) Representative density plots of IgD ( x ‐axis) and CD27 ( y ‐axis) status of B cells on days 0, 4, 7, 10, and 14. IgD + CD27 − B cells were classified as naïve B cells, and 97.1 % of B cells from baseline were IgD + CD27 − . Upon activation, B cells downregulate IgD and become IgD − CD27 − double negative B cells. CD27 (IgD − CD27 + ) was used as a marker for B‐cell differentiation into memory B cells and plasmablast subsets. Density plots representative of three experiments ( n = 3). ( C) CD95 MFI on B cells demonstrates how CD95 cannot be detected on the surface of naïve B cells (day 0) but is quickly upregulated upon stimulation. Fab20 completely inhibits CD95 upregulation. N = 3, bar graphs with whiskers equals mean ± SD. ( D) IgD MFI on B cells from the 14‐day assay. Day 0 B cells present with high IgD levels on the surface, but the expression is decreased to almost zero upon stimulation. Fab20 prevents IgD levels from decreasing, thus keeping B cells in a more naïve state. N = 3, bar graphs with whiskers equals mean ± SD. ( E) Immunoassay quantifying total IgG levels in supernatants from B cell cocultures on days 4, 7, 10, and 14 ( n = 4 from each timepoint, mean ± SD). On day 14, B‐cell differentiation caused untreated control and Fab isotype control B cells to produce high IgG levels. Fab20 completely prevented IgG production, also caused by the few surviving B cells on day 14. Unpaired t ‐tests of day 14 IgG levels, * p < 0.05, *** p < 0.01.

Article Snippet: Naïve human B cells were purified by negative selection from PBMCs using a human Naïve B cell isolation kit II (Miltenyi, 130‐091‐150) following the instructions given by the manufacturer.

Techniques: Activation Assay, Control, Labeling, Clinical Proteomics, Marker, Cell Differentiation, Expressing