B-cell helper–related markers and functional analysis of Tph cells in the blood of patients with cGVHD. Thawed mononuclear cell samples from patients with cGVHD were stained with multiple antibodies. (A) Cluster identification was visualized with UMAP, and (B) heatmap was generated with FlowSOM to analyze pooled data from 4 samples, 2 with no GVHD and 2 with active cGVHD. (C) Surface expression of PSGL1, ICOS, HLA-DR, and CCR2 were measured and calculated (N = 6). (D) Intracellular BCL6, MAF, and BLIMP1 were measured among naïve T cells, Tfh cells, and Tph cells between patients with no GVHD (N = 4) and patients with cGVHD (N = 6). (E) IL-21 production by Tfh and Tph cells from patients with no GVHD and patients with cGVHD was also tested (N = 5). (F) IL-2, CXCL13, IL-4, and interferon-γ expression among IL-21–producing Tfh and Tph cells were examined. (G) Sorted CD3⁺ CD4⁺ CD45RA^- PD1^hiCXCR5^- Tph and PD1^hiCXCR5⁺ Tfh cells were cultured with memory B cells in vitro in the presence of lipopolysaccharide and staphylococcal enterotoxin B with or without IL-21R Fc for 7 days. CD27⁺CD138⁺ plasma cells were measured by flow cytometry, and (H) plasma cell percentages were calculated. Supernatant IgG levels were measured by enzyme-linked immunosorbent assay (ELISA). Data represent mean ± SEM. P values were calculated by 2-way ANOVA with Holm-Sidak test (C, D, F) and 1-way ANOVA with Holm-Sidak test (H). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.