Regulatory T- and B-cell subsets in HSC transplanted RAG1^−/−mice. To identify regulatory T cells, splenocytes were stained for surface expression of CD3 and CD4 as well as intracellular FoxP3 expression. Representative graphs of individual stains for FoxP3 as well as quantification of FoxP3⁺ cells within the CD3⁺CD4⁺ population are shown in (A) and (B), respectively (Y, n = 8; O, n = 7; DY, n = 15; DO, n = 17). Regulatory T cells coexpressing the Ikaros zinc finger transcription factor Helios were identified within the FoxP3⁺ population. (C) Representative dot plot profiles of the underlying flow cytometric analysis and (D) quantification of Helios⁺ regulatory T cells within the FoxP3⁺ population (Y, n = 5; O, n = 5; DY, n = 4; DO, n = 7). For characterization of splenic B cells, CD19⁺ cells from young and old nontransplanted B6.SJL mice and RAG1^−/− recipients of DY and DO HSCs were stained for follicular B cells (FOB; CD21/35⁺CD23⁺), marginal zone B cells (MZ; CD21/35^highCD23⁻), and age-associated B cells (ABC; CD21/35⁻CD23⁻). Shown are representative graphs of individual stains (E) and quantification of these B-cell populations within the total CD19⁺ population (Y, n = 8; O, n = 7; DY, n = 13; DO, n = 16) (F). *P < .05; **P < .01; ***P < .001; ****P < .0001, 2-tailed unpaired Student's t-test, mean + SEM.