FIGURE 3. Human tBregs also express low levels of CD20.

Ex vivo generated human tBregs (from healthy donor B cells -treated with CM of MDA-MB-231 cells (17)) not only suppressed proliferation of human CD4⁺ and CD8⁺ T cells stimulated with anti-CD3 Ab (B-MDA, A), but also reduced cells expressing high levels of CD20 within CD81^hiCD25⁺ CD19⁺ cells (B). The suppressive activity (for CD4⁺ and CD8⁺ T cells) of human tBregs was retained within CD20^Low B cells (C), as shown by FACS sorting for CD20^Low B cells (enrichment >95%). Y-axis shows (%) Ki67⁺ CD4⁺ and CD8⁺ T cells ± SEM (C) of triplicate experiments. (D–F) Rituximab enriches for CD20^Low tBregs in patients with B-CLL, as shown by the increased presence of B cells expressing CD20^Low (broken line, D). In particular, compared with B cells before the treatment (continuous line, middle panel, D), the majority of B cells of patient 1 were CD20^Low after rituximab treatment (broken line, D). These CD20^Low cells efficiently suppressed proliferation of allogeneic CD4⁺ and CD8⁺ T cells (E) and induced FoxP3⁺ Treg conversion when mixed with CD25⁻ CD4⁺ T cells (% of FoxP3⁺ within CD4⁺, F). All data shown here were from triplicate experiments reproduced at least three times.