FIGURE 2. Depletion of CD20⁺ B cells after tumor challenge enriches for tBregs.

Anti-CD20 Ab efficiently depletes CD19⁺ cells in various organs (A) of 4T1.2 tumor-bearing BALB/c mice used in Figure 1D,E, but enriches for tBregs (CD81^hiCD25⁺ within CD19⁺ gated cells, B). CD19⁺ cells isolated from lymph nodes of tumor-bearing mice suppressed proliferation of syngeneic CD4⁺ and CD8⁺ T cells (C). In particular, B cells from anti-CD20 Ab –treated mice were much more suppressive for both CD4⁺ and CD8⁺ T cells (C). Proliferation was assessed using CFSE dilution assay of T cells stimulated with anti-CD3 Ab and incubated with equal numbers of B cells for 5 days. CD19⁺ cells from the spleen of tumor-bearing mice -treated with anti-CD20 Ab expressed low levels of surface CD20 as comaperd with naïve mice (Naïve, D) and non-treated tumor-bearing mice (Tumor-bearing, D). A representative FACS staining dot plots of CD20⁺ cells within CD19⁺ gated cells (D). Y-axis shows % of CD19⁺ cells (A), tBregs (B), or proliferated CD4⁺ and CD8⁺ T cells (C) ± SEM of four-eight mice per group experiments reproduced at least three times.