Excess BAFF after allo-BMT is associated with increased BCR-activated circulating and lesional tissue GL7⁺ B cells in cGVHD. (A) Relative response to BCR agonist by B cells from mice with (BM + Sp) vs without cGVHD manifestations (BM only and Syn). Five days after ex vivo BCR stimulation with anti-IgM (10 µg/mL), BCR activation was measured by pregating on live 7-AAD⁻CD19⁺ splenic B cells in CFSE dilution assay. Activated dividing cells dilute CFSE as they proliferate. Data were pooled from 3 separate experiments performed on splenocytes from mice euthanized at the end of BMT experiments (on days 83, 88, and 98 post-BMT); n = 6 (BM only), n = 9 (BM + Sp), and n = 3 (Syn). (B) Proportion of phosphorylated SYK (pSYK) in B cells from cGVHD mice compared with control mice after ex vivo BCR stimulation. Shown are the relative frequencies of pSYK⁺ B cells after pregating on total B cells or pSYK⁺ B cells of the GL7⁻ vs GL7⁺ B-cell subsets. Peripheral blood was taken from mice on day 54 after allo-BMT, red blood cells were lysed, and peripheral blood mononuclear cells were stimulated with anti-IgM (10 µg/mL) for 5 minutes before fixation and phosphoflow staining for flow cytometric analysis; n = 5 of each group. (C) Relative cell surface expression of IgM on total CD19⁺ B cells and GL7⁺ B cells. Representative flow cytometric histograms are shown after staining for cell surface membrane–bound IgM on total B cells (top panel) or on the GL7⁺ B-cell subset (bottom panel) in peripheral blood of cGVHD (BM + Sp) mice vs no cGVHD (BM only) mice at day 42 post-BMT. Cells were pregated on Zombie⁻CD19⁺ cells (top panel) or pregated on Zombie⁻CD19⁺GL7⁺ cells (bottom panel). (D) BAFF together with BCR stimulation promotes GL7 expression. Proportion of GL7⁺ B cells after treatment with surrogate antigen with or without exogenous recombinant BAFF was evaluated. B cells isolated from the blood of naïve mice were treated with 5 µg/mL of anti-IgM or vehicle control overnight (18 hours) with or without additional BAFF (5 ng/mL) before the frequency of Zombie⁻CD19⁺GL7⁺ B cells was measured by flow cytometry; n = 5. (E) Circulating GL7⁺ B-cell frequencies over time after allo-BMT in cGVHD (BM + Sp) mice compared with no cGVHD control allo-BMT (BM only) mice and Syn control mice. Representative flow cytometric profiles were from analysis of peripheral blood on day 42 post-BMT. Numbers represent the percentages of GL7⁺ B cells after pregating on Zombie⁻CD19⁺ live cells. Data were pooled from 2 experiments; n = 15 (BM only), n = 9 (BM + Sp), and n = 5 (Syn). (F) Eye scores in diseased (BM + Sp) vs nondiseased (BM only) mice at day 35 after allo-BMT, assessed in a masked fashion. Representative photographs taken under ×3.2 magnification using a Zeiss Stemi 2000-C stereo microscope with Nikon Coolpix P5100 camera. Eye scores were calculated by adding scores for the following related manifestations: chemosis, mucoid discharge, and corneal opacity; n = 15 of each group. (G) Proportion of GL7⁺ cells in parent gate of CD19⁺ B cells by flow cytometric analysis of cells isolated from conjunctival tissue samples from mice with or without cGVHD at day 42 or 43 after allo-BMT. This result was combined from 2 experiments; n = 8 of each group. Statistical analysis was performed by Kruskal-Wallis test (A,E), unpaired Student t test with Welch’s correction (B,G), ordinary 1-way analysis of variance with Tukey’s multiple comparisons test (D), or Mann-Whitney test (F) using GraphPad Prism 8 software. *P < .05, **P < .01, ***P < .001, ****P < .0001. NS, not significant (P > .05).