Figure 7.
Persistent excess BAFF after allo-BMT promotes cGVHD manifestations, GL7+ B cells, and antirecipient antibody production. (A) Ocular cGVHD manifestations in recipients of T cell–depleted WT BM, BAFF Tg BM, or Syn-BMT. Representative photographic examples of eye pathology via Zeiss Stemi 2000-C stereo microscope with Nikon Coolpix P5100 camera (×3.2 magnification) are shown above the graph of the compiled cGVHD eye pathology scores. Eye scores include chemosis, mucoid discharge, and corneal opacity as assessed by biomicroscopic examination by a masked investigator at day 35 after allo-BMT. Data shown are from 1 representative experiment of 3 independent repeats; n = 10 each group (BM only and Syn groups). For reference, scores for the T cell–replete BAFF Tg BM + Sp are shown in the column on the far left; n = 9 (BAFF Tg BM + Sp). (B) Relative body weights in recipients of BM only T cell–depleted BAFF-Tg vs WT donor cells vs Syn BAFF Tg BM only cells; n = 5 (Syn-BMT) and n = 10 for other groups. This experiment was repeated, and data shown are representative of 2 experiments. (C) Frequency of GL7+ peripheral blood B cells in recipients of T cell–depleted WT BM, BAFF Tg BM, or Syn-BMT as assessed by flow cytometry on day 61 after allo-BMT. We quantified the proportion of GL7+ cells after pre-gating on Zomibie−CD19+ live cells in blood from allo-BMT mice using T cell–depleted BAFF Tg BM only vs WT BM only cells or Syn BAFF Tg BM only. This experiment was repeated 3 times, with each producing similar results; n = 5 (BAFF Tg BM + Sp) and n = 10 for all other groups. Similar results (data not shown) were also found on day 137 after allo-BMT in the above experiment. (D) Representative immunofluorescent images showing the relative amounts of plasma IgG taken after BMT bound to salivary gland tissue sections from BALB/c RAG−/− mice. Donor-derived antirecipient IgG after allo-BMT from recipient of T cell–depleted BAFF Tg BM ± Sp vs T cell–depleted WT BM only transplants and Syn BAFF Tg BM only is detected with an anti-mouse IgG- Alexa Fluor 488 secondary antibody. Images were taken by Zeiss Axiovert 200M fluorescence microscope with ApoTome using 200× lenses. (E) Relative fluorescence intensity of the plasma IgG-stained BALB/c RAG−/− salivary gland tissue sections shown in panel D quantitated using ImageJ software. The integrated intensity value measured by ImageJ per image field is shown as mean fluorescence intensity. Plasma from 2 mice of each group was assessed, with 2 to 3 images taken from each slide. Each symbol represents fluorescence intensity of a single image. This quantification strategy was repeated twice. Statistical analysis was performed by Kruskal-Wallis test (A), repeated measures analysis of variance (ANOVA) of body weight data from day 20 to 70 (B), or ordinary 1-way ANOVA with Tukey’s multiple comparisons test (C-E) using GraphPad Prism 8 software. *P < .05, **P < .01, ***P < .001. NS, not significant (P > .05).

Persistent excess BAFF after allo-BMT promotes cGVHD manifestations, GL7+ B cells, and antirecipient antibody production. (A) Ocular cGVHD manifestations in recipients of T cell–depleted WT BM, BAFF Tg BM, or Syn-BMT. Representative photographic examples of eye pathology via Zeiss Stemi 2000-C stereo microscope with Nikon Coolpix P5100 camera (×3.2 magnification) are shown above the graph of the compiled cGVHD eye pathology scores. Eye scores include chemosis, mucoid discharge, and corneal opacity as assessed by biomicroscopic examination by a masked investigator at day 35 after allo-BMT. Data shown are from 1 representative experiment of 3 independent repeats; n = 10 each group (BM only and Syn groups). For reference, scores for the T cell–replete BAFF Tg BM + Sp are shown in the column on the far left; n = 9 (BAFF Tg BM + Sp). (B) Relative body weights in recipients of BM only T cell–depleted BAFF-Tg vs WT donor cells vs Syn BAFF Tg BM only cells; n = 5 (Syn-BMT) and n = 10 for other groups. This experiment was repeated, and data shown are representative of 2 experiments. (C) Frequency of GL7+ peripheral blood B cells in recipients of T cell–depleted WT BM, BAFF Tg BM, or Syn-BMT as assessed by flow cytometry on day 61 after allo-BMT. We quantified the proportion of GL7+ cells after pre-gating on Zomibie−CD19+ live cells in blood from allo-BMT mice using T cell–depleted BAFF Tg BM only vs WT BM only cells or Syn BAFF Tg BM only. This experiment was repeated 3 times, with each producing similar results; n = 5 (BAFF Tg BM + Sp) and n = 10 for all other groups. Similar results (data not shown) were also found on day 137 after allo-BMT in the above experiment. (D) Representative immunofluorescent images showing the relative amounts of plasma IgG taken after BMT bound to salivary gland tissue sections from BALB/c RAG−/− mice. Donor-derived antirecipient IgG after allo-BMT from recipient of T cell–depleted BAFF Tg BM ± Sp vs T cell–depleted WT BM only transplants and Syn BAFF Tg BM only is detected with an anti-mouse IgG- Alexa Fluor 488 secondary antibody. Images were taken by Zeiss Axiovert 200M fluorescence microscope with ApoTome using 200× lenses. (E) Relative fluorescence intensity of the plasma IgG-stained BALB/c RAG−/− salivary gland tissue sections shown in panel D quantitated using ImageJ software. The integrated intensity value measured by ImageJ per image field is shown as mean fluorescence intensity. Plasma from 2 mice of each group was assessed, with 2 to 3 images taken from each slide. Each symbol represents fluorescence intensity of a single image. This quantification strategy was repeated twice. Statistical analysis was performed by Kruskal-Wallis test (A), repeated measures analysis of variance (ANOVA) of body weight data from day 20 to 70 (B), or ordinary 1-way ANOVA with Tukey’s multiple comparisons test (C-E) using GraphPad Prism 8 software. *P < .05, **P < .01, ***P < .001. NS, not significant (P > .05).

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