Tumor microenvironment differences between diagnostic and relapsed classic Hodgkin lymphoma revealed by scRNA-seq

Classical Hodgkin lymphoma (CHL) is characterized by a complex tumor microenvironment (TME) that supports disease progression. Although immune cell recruitment by Hodgkin and Reed-Sternberg (HRS) cells is well documented, the role of nonmalignant B cells in relapse remains unclear. Using single-cell RNA sequencing (scRNA-seq) on paired diagnostic and relapsed CHL samples, we identified distinct shifts in B-cell populations, particularly an enrichment of naïve B cells and a reduction of memory B cells in early-relapse CHL compared to late-relapse and newly diagnosed CHL. The enrichment of naïve B cells in early relapse biopsies was confirmed in independent validation cohorts using scRNA-seq and immunohistochemistry. Notably, naïve B cells in early-relapse samples exhibited high expression of LGALS9, an immunosuppressive gene encoding galectin-9, which binds to HAVCR2 (T-cell immunoglobulin and mucin domain-containing protein 3 [TIM-3]) on regulatory T cells (Tregs). Cell-cell interaction analysis revealed the importance of interactions between LGALS9+ naïve B cells and HAVCR2+ Tregs in the early-relapse setting. Spatial analysis by imaging mass cytometry confirmed close proximity of galectin-9-positive naïve B cells with TIM-3+ CD4+ T cells and HRS cells, pointing to their role in shaping an immunosuppressive niche. Our findings highlight a previously unrecognized population of galectin-9-positive naïve B cells with immunoregulatory potential in early-relapse CHL and provide new insights into the spatial and transcriptional architecture of the relapsed TME in CHL.