Background: Over the past decade knowledge about the mechanisms by which the neoplastic Hodgkin and Reed-Sternberg (HRS) cells recruit and manipulate the extensive tumor microenvironment (TME) of classical Hodgkin lymphoma (cHL) has significantly expanded. While some findings support the concept that characteristics of the host immune system contribute to the composition of the TME, there is increasing evidence that somatically acquired genetic alterations and phenotypes of HRS cells influence disease evolution and underlie specific TME interactions. Integrating these concepts, the cHL TME might be the reflection of a symbiotic ecosystem containing the malignant cells and host immune cells. Dissection of this symbiotic relationship at single-cell resolution will be critical for a better understanding of cHL pathogenesis with relevance for improved therapeutic targeting.

Knowledge Gained: We present single-cell transcriptome sequencing data derived from cell suspensions of 22 Hodgkin lymphoma patients (representative of the nodular sclerosis and mixed cellularity subtype) that confirmed the previously identified predominance of Th1 polarized T regulatory cells in the TME. In comparison to 5 reactive lymph node samples, we describe a highly enriched, HL-specific T-cell subset that is characterized by high LAG3 expression and an immunosuppressive cytokine profile, consistent with type 1 regulatory (Tr1) T cells. Analyzing co-expression of inhibitory T-cell makers on the single-cell level, LAG3 expression occurred mostly in a mutually exclusive pattern to the canonical Treg marker FOXP3. Furthermore, using imaging mass cytometry and multicolor immunohistochemistry, we revealed a spatial pattern of rosetting LAG3+ cells in the direct vicinity of MHC-II negative HRS cells, suggesting differential microenvironment architecture dependent on malignant cell phenotypes. Comparative analysis of LAG3 expression across a spectrum of B-cell lymphomas confirmed the high abundance of these cells in cHL, but also suggest that LAG3+ CD4+ and LAG3+ CD8+ T-cell subsets play a role in the related entity of primary mediastinal large B-cell lymphoma (PMBCL) and diffuse large B-cell lymphoma (DLBCL). Expanding our studies to the lymphocyte-rich (LR) subtype of cHL, we also revealed subtype-specific differences in background B cell and T follicular helper (TFH) cell content, suggesting an important role of the CXCR5-CXCL13 axis in cHL.

Significance: Characterization of the TME using multiparametric single-cell technologies, including multicolor immunohistochemistry, flow-based mass cytometry, imaging mass cytometry, single-cell RNAseq, and proximity ligation assays, has provided unprecedented insight into HL biology. These studies fuel hope for accelerated development of immunotherapies targeting the TME and predictive biomarker development that might guide the appropriate selection of checkpoint inhibitors.

Citation Format: Christian Steidl. The immune cell microenvironment of classical Hodgkin lymphoma by single-cell analysis [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA04.