Single-cell profiling reveals the importance of CXCL13/CXCR5 axis biology in lymphocyte-rich classic Hodgkin lymphoma

Lymphocyte-rich classic Hodgkin lymphoma (LR-CHL) is a rare subtype of Hodgkin lymphoma. Recent technical advances have allowed for the characterization of specific cross-talk mechanisms between malignant Hodgkin Reed-Sternberg (HRS) cells and different normal immune cells in the tumor microenvironment (TME) of CHL. However, the TME of LR-CHL has not yet been characterized at single-cell resolution. Here, using single-cell RNA sequencing (scRNA-seq), we examined the immune cell profile of 8 cell suspension samples of LR-CHL in comparison to 20 samples of the mixed cellularity (MC, 9 cases) and nodular sclerosis (NS, 11 cases) subtypes of CHL, as well as 5 reactive lymph node controls. We also performed multicolor immunofluorescence (MC-IF) on tissue microarrays from the same patients and an independent validation cohort of 31 pretreatment LR-CHL samples. ScRNA-seq analysis identified a unique CD4(+) helper T cell subset in LR-CHL characterized by high expression of Chemokine C-X-C motif ligand 13 (CXCL13) and PD-1. PD-1(+)CXCL13(+) T cells were significantly enriched in LR-CHL compared to other CHL subtypes, and spatial analyses revealed that in 46% of the LR-CHL cases these cells formed rosettes surrounding HRS cells. MC-IF analysis revealed CXCR5(+) normal B cells in close proximity to CXCL13(+) T cells at significantly higher levels in LR-CHL. Moreover, the abundance of PD-1(+)CXCL13(+) T cells in the TME was significantly associated with shorter progression-free survival in LR-CHL (P = 0.032). Taken together, our findings strongly suggest the pathogenic importance of the CXCL13/CXCR5 axis and PD-1(+)CXCL13(+) T cells as a treatment target in LR-CHL.