High soluble BAFF levels are associated with an altered peripheral B-cell compartment and low BAFF-R detectability on B cells in mice with cGVHD manifestations. (A) Calculated BAFF/B-cell ratios over time after BMT in mice that develop (BM + Sp) vs do not develop cGVHD manifestations (BM only and Syn). BAFF levels shown in Figure 1A and peripheral blood B-cell numbers (supplemental Figure 3A) were used to calculate BAFF per B-cell ratios as shown. Data are representative of 3 repeats; n = 10 (BM + Sp and BM only) and n = 5 (Syn). Arrow indicates the median time of onset of ocular GVHD manifestations day 30 post-BMT.⁴¹  (B) Relative proportion of transitional B cells in peripheral blood from mice that develop (BM + Sp) vs those that do not develop cGVHD (BM only and Syn). Total CD19⁺CD93⁺ transitional B cells in peripheral blood were assessed by flow cytometry after allo-BMT. Day 42 data shown are combined from 4 independent experiments; n = 5 of each group at days 20, 61, and 82; n = 20 (BM only), n = 18 (BM + Sp), and n = 5 (Syn) at day 42. (C) Representative contour plot of flow cytometric data of pregated CD93⁺ B cells examined for IgM and CD23 staining. Frequency of transitional 3 (T3) B cells from the blood of cGVHD mice (BM + Sp) compared with controls (BM only and Syn) is shown on day 42 post-BMT. T3 cells were identified by gating on CD19⁺CD93⁺IgM^lowCD23^high cells (shown on left panels as contour plots); numbers are percentage of T3 cells. Data shown are combined data from 3 separate experiments; n = 13 (BM only), n = 9 (BM + Sp), and n = 5 (Syn). (D) BAFF-R on the surface of B cells from the blood of mice with (BM + Sp) or without cGVHD (BM only and Syn) on days 34 to 36 after allo-BMT. BAFF-R staining using anti–BAFF-R antibody (clone 7H22-E16; Biolegend) of whole blood was performed using flow cytometry by pregating on 7-AAD⁻CD19⁺ live B cells before median fluorescence intensity of BAFF-R was determined. Data shown were pooled from 2 separate experiments; n = 21 (BM only), n = 15 (BM + Sp), and n = 10 (Syn includes both Syn BM only, n = 5 and Syn BM + Sp, n = 5 combined). (E) Examination of BAFF-R on BAFF Tg B cells for BAFF occupancy using BAFF dissociation and acid elution methods. Top panel shows the BAFF dissociation assay that was performed as follows. Splenocytes of BAFF Tg mice were incubated for 2.5 hours in 4 mL of RPMI complete medium at 37°C and pipetted every 30 minutes. Bottom panel shows the acid elusion assay that was performed as follows. Peripheral blood cells from BAFF Tg mice were pelleted after red blood cells were lysed, quickly suspended in citrate buffer (pH, 2.4) for 1 minute at room temperature, and then immediately diluted in PBS to stop the reaction. BAFF-R expression was assessed by flow cytometric analysis, with pregating on CD19⁺Zombie⁻ live cells. Dashed line represents the isotype control histogram. Solid lines indicate BAFF-R–stained cells. Statistical analysis was performed by Kruskal-Wallis test (A-D) using GraphPad Prism 8 software. *P < .05, **P < .01, ***P < .001, ****P < .0001. NS, not significant (P > .05).