Mononuclear cells from human tonsil or peripheral blood were stained with mouse monoclonal antibodies against human CD19, IgD, CD27 and CD38, and CD19⁺ cells were gated for analysis. The nomenclatures for B cell subsets defined by each scheme are also depicted.

The frequency of IgD⁺CD27⁺ unswitched memory cells in the CD19⁺ PBL from a cohort of normal subjects (n=42) and SLE patients (n=35) was determined, and was found to be significantly higher in SLE patients (12.7±4.7%) than in normal subjects (5.6±4.0%) (p<0.0001). In some of the cohorts, the expression levels of CD1c in the various subsets defined by IgD/CD27 were also analyzed, and the fraction of CD1c-expressing cells was substantially decreased in CD27⁺ subsets in SLE patients. Graphs shown are the representatives of the staining profiles of normal subjects and SLE patients.

The fractions of IgD⁻CD27⁺ switched memory and IgD⁺CD27⁺ unswitched memory cells in the CD19⁺ PBL were determined from a cohort of SLE patients before and after rituximab treatment. Pre-treated patients displayed an expansion of switched memory and an accompanying reduction of unswitched memory B cells, compared to healthy controls. Patients with shorter clinical responses to rituximab exhibited memory profiles after reconstitution similar to the pre-treated group. Patients with prolonged clinical remissions experienced a delay in both switched and unswitched memory B cell reconstitution.

(A) In contrast to healthy subjects, memory B cells were predominantly found in the early Bm5 compartment of the tonsil in SLE patients. After BCDT and reconstitution, the majority of memory B cells were found in the late Bm5 compartment, and the FcRH4⁺CD27⁻ cells in the whole Bm5 compartment were drastically reduced. (B) RA patients treated with anti-TNFα exhibited decreases in the IgD⁻CD27⁺ switched and IgD⁺CD27⁺ unswitched memory B cell populations in the peripheral blood, compared to the healthy control and RA patients treated with methotrexate

(A) Our previous results demonstrated an expansion of IgD⁻CD27⁻ DN memory cells in the periphery of active SLE patients (12). Graphs show the representative IgD/CD27 expression profiles from a normal subject and an active SLE patient. (B) Seven healthy subjects were inoculated with RSV A2 at day 0, and the fractions of IgD⁻CD27⁻ DN memory cells in the peripheral blood B cells were followed for a period of up to 28 days. The expansion of IgD⁻CD27⁻ cells occurred as early as 4 days after RSV infection and peaked by day 8 for all but one subject, for whom the expansion was observed at day 12. By day 28, the levels of DN cells waned to the pre-infection levels. Although the antigen specificities of these expanded DN memory cells remain to be elucidated, the waxing and waning of these cells in patients exposed to RSV suggests a role for these cells in protective memory responses. Each line in the graph represents an individual subject.

Ten-color flow cytometry panels were developed to stain the mononuclear cells from peripheral blood (A, B) and tonsil (C) with antibodies against human CD19, CD3, IgD, IgM, IgG, CD1c, CD10, CD24, CD27, CD38, CD45(B220), and FcRH4. Cells were also stained with LIVE/DEAD Fixable Aqua Dead Cell Stain. Live CD19⁺ B cells were gated for analysis. (A) IgD⁻CD27⁺ switched memory, IgD⁻CD27⁻ DN memory, Bm3+4, early Bm5 and late Bm5 cells (blue dots) from the peripheral blood were superimposed on the total CD19⁺ B cells (red dots) to demonstrate where these subsets defined by one classification scheme would fall under other classification schemes. (B) The expression levels of CD24 and B220 in the IgM and IgG fractions of IgD⁻CD27⁺ and IgD⁻CD27⁻ memory cells in the periphery were depicted. IgG memory cells, in contrast to IgM memory cells, exhibited heterogeneous expression levels of CD24 and B220. (C) The expression levels of CD27, B220 and FcRH4 in the early Bm5, late Bm5 and Bm5 as a whole from the tonsil were depicted in various bivariate plots, which revealed the heterogeneity of these memory B cells.