Antibody-producing donor B cells augment lymphoid tissue damage and loss of GCs in cGVHD recipients. HCT was carried out with BALB/c recipients as described in Figure 1. (A) Kinetic changes in donor CD4⁺ T-cell expansion in the spleen and lymph nodes and infiltration in the GVHD target tissues (skin, liver, and lung) at 40 and 60 days after HCT. Spleen, PLNs, skin, liver and lung of recipients were harvested for analysis of donor CD4⁺ T yield. Means ± SE of the yield of CD5.1⁺TCRβ⁺CD4⁺ T cells are shown. N = 4 from at least 2 replicate experiments. (B,E) Frozen spleen sections from TCD-BM-no-cGVHD, WT-cGVHD, IgH^μγ1-no-cGVHD, and IgH^μγ1-transient-cGVHD recipients at days 40 and 60 after HCT were analyzed by immunofluorescent staining of GC structures. Spleen sections were stained for B220 (green), CD3 (red), peanut agglutinin (PNA; blue), 40 (B) and 60 (E) days after HCT. The blue area in the merged picture is the GC. A photomicrograph (original magnification, ×200) representative of 4 replicate experiments is shown. (C,F) The size of the GC at days 40 and 60 after HCT was quantified by measuring the area of PNA staining in Photoshop C3, and the frequencies of GCs at days 40 and 60 after HCT were quantified by counting the number of GCs per mm² of spleen section. Means ± SE of the size and frequency of GCs are shown. N = 4 from at least 2 replicate experiments. (D,G) Frequency of T_FH cells (defined as PD-1^hiCXCR5⁺CD4⁺ T cells) and GC B cells (defined as GL7⁺Fas⁺ B cells). On days 40 (D) and 60 (G) after HCT, splenocytes were stained with anti-CD4, T-cell receptor β (TCRβ), programmed cell death 1 (PD-1), and CXC chemokine receptor 5 (CXCR5) monoclonal antibodies (mAbs), and gated CD4⁺TCRβ⁺ cells are shown as PD-1 vs CXCR5. Splenocytes were also stained with anti-CD19, GL7, and Fas mAbs, and gated CD19⁺ cells are shown as Fas vs GL7. CXCR5⁺PD-1⁺ T_FH and Fas⁺GL7⁺ GC B cells are gated. Representative staining profiles are shown. Means ± SE of the frequency of T_FH cells and GC B cells are shown. N = 4 from at least 2 replicate experiments (*P < .05, **P < .01, ***P < .001, ****P < .0001).