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

• Naïve B cells expressing LGALS9 (Galectin-9) are distinctly increased in early-relapse CHL compared to diagnostic and late-relapse samples.

• Galectin-9+ naïve B cells engage with TIM-3⁺ T cells, potentially contributing to an immuno-suppressive TME in early-relapse CHL.

Classical Hodgkin Lymphoma (CHL) is characterized by a complex tumor microenvironment (TME) that supports disease progression. While immune cell recruitment by Hodgkin and Reed-Sternberg (HRS) cells is well-documented, the role of non-malignant 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 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 (TIM-3, T-cell immunoglobulin and mucin domain-containing protein 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+ 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+ 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.