A, CD19^hi and CD19^lo B cells were sorted (using gates illustrated in Fig. 1B) from four patients with differing anti-Sm titers, and ELISpot used to determine the number of anti-Sm IgG ASCs in each group with and without stimulation by pansorbin, anti-CD40, IL-10 and IL-2. B, Anti-Sm ASCs per million CD19^hi or CD19^lo B cells in each of four patients. Patient 1 did not have detectable serum anti-Sm titer, and no anti-Sm ASCs were seen in either cell group. C, Anti-Sm ASCs per million cells in each group vs. log₁₀[anti-Sm serum titer], showing an exponential relationship between the number of anti-Sm ASCs in the CD19^hi B cell group and the serum titer of anti-Sm in the corresponding patient.

A and B, Transcription factor expression in indicated sorted B cell groups as determined by RQ RT-PCR. Each bar represents a different individual. Gating for cell sorting is shown in Fig. 1B. A, Relative expression in CD19^hi B cells as compared to CD19^lo B cells from the same patient. B, Relative expression in CD19⁺CD38⁻IgD⁻ memory B cells as compared to all other CD19⁺ B cells from the same healthy control. C, Representative histograms for Ki-67 staining in non-lymphoid PBMCs (shaded) and CD19^lo (thin line) or CD19^hi (thick line) patient PB B cells and for PI staining of indicated subsets. Percentages indicate cells falling into drawn gate.

A, Clinical response of CD19^hi SLE patients and CD19^lo SLE patients to rituximab treatment. Response was defined as an improvement in the SLAM or SLEDAI score of at least 3 points and duration of response defined as the time point at which these disease activity indexes increased by 3 points or an increase in steroid dosage was required. NR=no response; OG=ongoing response. B, Representative histograms showing gating for CD19^hi and CD19^lo B cells from an SLE patient. Only CD19⁺ PB B cells are included in this gate. The bottom histogram shows CD20 expression by CD19^hi and CD19^lo B cells and healthy control B cells (all CD19⁺ B cells). Results are representative of 3 patients.

PB cells were permeabilized and stained using antibodies recognizing the indicated molecules. Gating for CD19^hi and CD19^lo B cells is shown in Fig. 1B. A, Fold increase in MFIs of CD19 or phosphorylated CD19 (pCD19) in CD19^hi vs CD19^lo cells. B, Representative histograms of PB cells from a CD19^hi patient and healthy control (HC) stained for CD19 and pSyk, pERK1/2, or an isotype control. Gating is on all CD19⁺ B cells. C, Representative single parameter histograms showing staining for phosphorylated CD19 and phosphorylated or total Syk and ERK1/2 in PB B cells from a healthy control (shaded) and CD19^lo (thin line) and CD19^hi (thick line) B cells from the same patient. Gating is on CD19^hi and CD19^lo B cells as shown in Fig. 1B. D, Fold increases in MFI for the labeled signaling molecules in CD19^lo vs. CD19^hi cells from the same patient. A and D, Each dot represents a different patient and/or day. At least four different patients are included for each signaling molecule. * p≤0.05; **p≤0.001.

A, Representative histograms of PB B cells from a CD19^hi patient and a healthy control after incubation with media alone or with anti-IgG for 10 min. Histograms are gated on CD19⁺ PB B cells and the percentages give are of B cells falling within each indicated gate. B, Representative single parameter histograms for each of the indicated phosphorylated molecules in PB B cells from a healthy control and CD19^lo and CD19^hi B cells from a patient after incubation with media (shaded) or pansorbin (line) for 10 min. Gating is illustrated in Fig. 1B. C, Fold change in the percentage of cells falling into the positive gate for each of the phosphorylated signaling molecules tested in cells incubated with media or pansorbin in a healthy control or in CD19^lo and CD19^hi B cells from the same patient. Each point represents a different individual. D, CD19^hi and CD19^lo B cells (see Fig. 2A) were sorted and IgG antibody secreting cells (ASCs) determined after incubation with either media alone or with pansorbin, anti-CD40, IL-10 and IL-2 for 6 days. Each dot represents a different patient. * p≤0.05; **p≤0.001.

A and B, CXCR expression in indicated sorted B cell groups as determined by RQ RT-PCR. Each bar represents a different individual. A, Relative expression in CD19^hi B cells as compared to CD19^lo B cells from the same patient. B, Representative histogram of CD19⁺ B cells from a healthy control showing gating of memory B cells (circled; CD38-, IgD-). The non-memory B cells used were gated as all CD19⁺ B cells that fell outside this gate. Bars represent relative transcript expression in CD19⁺CD38⁻IgD⁻ memory B cells as compared to all other CD19⁺ B cells from the same healthy control. C, Surface CXCR3 expression determined by flow cytometry on healthy control B cells (shaded) or in CD19^lo (thin line) or CD19^hi (thick line) B cells from the same patient. Histogram is representative of three patients. D, Chemotaxis of CD19^hi B cells in response to the CXCR3 ligand CXCL9, the CXCR4 ligand CXCL12, or media alone. Migration was determined by collection, staining, and flow cytometry of cells which migrated into lower wells after 90 minutes. No chemotaxis was observed in response to CXCL12 and media alone by cells from patient 2.