Splenic T cells are not activated in IgH-hEBI2 mice, but increased levels of the B1 B-cell–secreted IL-10 are observed. (A) CD4⁺ and CD8⁺ T cells were depleted by injection of antibodies against CD4, CD8, and Thy1. The percentage of CD4⁺ and CD8⁺ cells in WT and IgH-hEBI2 mice were measured by FACS. The results are given as percentage of total spleen cells and are mean ± SEM of data from 6 T-cell–depleted IgH-hEBI2 and WT mice and 3 nondepleted control mice. (B) Spleen weight of WT and IgH-hEBI2 mice after 4 weeks of T-cell–depleting injections with antibodies against CD4, CD8, and Thy1. The results are given as mean ± SEM of data from 6 T-cell–depleted IgH-hEBI2 and WT mice. (C) Gating scheme for activated T cells. (D) Spleen cells from 12- to 15-week-old WT and IgH-hEBI2 mice were analyzed for the presence of the T-cell activation marker CD69 by FACS. Numbers indicate the percentage of cells falling into each gate as shown in panel C. The results are mean ± SEM of data from 5 WT and 6 IgH-hEBI2 mice. (E-F) qPCR analysis of IL-4, IL-10, and IL-21 expression in spleen cells from young (12-15 weeks) (E) and old (16-18 months) (F) IgH-hEBI2 and WT mice relative to the expression of the control gene GAPDH. The results are mean ± SEM of data from 4 mice. **P < .01 and ***P < .001 by the nonparametric Mann-Whitney test. (G) qPCR analysis of IL-10 expression in CD19-enriched and -depleted spleen samples from 12- to 15-week-old IgH-hEBI2 and WT mice relative to the expression of the control gene, GAPDH. The results are mean ± SEM of data from 4 mice. *P < .05 by the nonparametric Mann-Whitney test.