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Reconstitution of B-cells over time. To characterize B-cell subsets, peripheral blood mononuclear cells were stained with fluorochrome-conjugated monoclonal antibodies (BD Biosciences) directed against the following antigens: CD45 (V500-C), CD3 (APC), CD19 (APC-H7), CD27 (BV421), IgD (PE), CD38 (BV711), CD4 (BV605), and CD8 (PE), as well as 7-AAD (Miltenyi Biotech). The stained cells were then analyzed using multicolor flow cytometry (BD FACS Lyric). The subsets of gated CD19 + cells were identified based on surface marker expression as follows: naïve (CD19+CD27-IgD+), nonswitched memory (CD19+CD27+IgD+), switched memory (CD19+CD27+IgD-), and double negative (CD19+CD27-IgD-); and within the CD19+CD38++ population, we distinguished transitional cells (CD19+CD38++CD27-IgD+), plasmablasts (CD19+CD38++CD27+IgD-), and double-negative CD38 + cells (CD19+CD38++CD27-IgD-). B-cell subsets were expressed as a percentage of the total lymphocyte count. (a) B-cell subpopulations were assessed in 39 patients at baseline and in 36 patients at 3 (M3), 6 (M6), 9 (M9), 12 (M12), and 18 (M18) months following the first rituximab infusion. The 3 patients who did not receive treatment were excluded from the follow-up. Complete depletion of B-cells was observed in all patients at M3 post-rituximab for all B-cell subpopulations. CD19 + cells reappeared 6 months after rituximab infusion. Naive cells re-emerged the most among B-cells, followed by CD38 + cells, transitional cells and finally memory cells. Data are shown as mean values (dots). (b–k) B-cell subpopulations at baseline and at subsequent time points were compared between relapsing patients ( n = 8) and nonrelapsing patients ( n = 19); the 2 patients who received additional anti-CD20 infusions were excluded from subsequent analyses. Data are shown as medians and interquartile range (IQR). P -values were calculated by comparing the median values of each cell subpopulation between relapsing and nonrelapsing patients using a nonparametric, unpaired Mann–Whitney U test.

Journal: Kidney International Reports

Article Title: Early-Stage B-cells Predict Relapse After Rituximab Treatment in Patients With Membranous Nephropathy

doi: 10.1016/j.ekir.2026.106365

Figure Lengend Snippet: Reconstitution of B-cells over time. To characterize B-cell subsets, peripheral blood mononuclear cells were stained with fluorochrome-conjugated monoclonal antibodies (BD Biosciences) directed against the following antigens: CD45 (V500-C), CD3 (APC), CD19 (APC-H7), CD27 (BV421), IgD (PE), CD38 (BV711), CD4 (BV605), and CD8 (PE), as well as 7-AAD (Miltenyi Biotech). The stained cells were then analyzed using multicolor flow cytometry (BD FACS Lyric). The subsets of gated CD19 + cells were identified based on surface marker expression as follows: naïve (CD19+CD27-IgD+), nonswitched memory (CD19+CD27+IgD+), switched memory (CD19+CD27+IgD-), and double negative (CD19+CD27-IgD-); and within the CD19+CD38++ population, we distinguished transitional cells (CD19+CD38++CD27-IgD+), plasmablasts (CD19+CD38++CD27+IgD-), and double-negative CD38 + cells (CD19+CD38++CD27-IgD-). B-cell subsets were expressed as a percentage of the total lymphocyte count. (a) B-cell subpopulations were assessed in 39 patients at baseline and in 36 patients at 3 (M3), 6 (M6), 9 (M9), 12 (M12), and 18 (M18) months following the first rituximab infusion. The 3 patients who did not receive treatment were excluded from the follow-up. Complete depletion of B-cells was observed in all patients at M3 post-rituximab for all B-cell subpopulations. CD19 + cells reappeared 6 months after rituximab infusion. Naive cells re-emerged the most among B-cells, followed by CD38 + cells, transitional cells and finally memory cells. Data are shown as mean values (dots). (b–k) B-cell subpopulations at baseline and at subsequent time points were compared between relapsing patients ( n = 8) and nonrelapsing patients ( n = 19); the 2 patients who received additional anti-CD20 infusions were excluded from subsequent analyses. Data are shown as medians and interquartile range (IQR). P -values were calculated by comparing the median values of each cell subpopulation between relapsing and nonrelapsing patients using a nonparametric, unpaired Mann–Whitney U test.

Article Snippet: To characterize B-cell subsets, peripheral blood mononuclear cells were stained with fluorochrome-conjugated monoclonal antibodies (BD Biosciences) directed against the following antigens: CD45 (V500-C), CD3 (APC), CD19 (APC-H7), CD27 (BV421), IgD (PE), CD38 (BV711), CD4 (BV605), and CD8 (PE), as well as 7-AAD (Miltenyi Biotech).

Techniques: Staining, Bioprocessing, Flow Cytometry, Marker, Expressing, MANN-WHITNEY