Single-Cell Transcriptome Analysis Reveals Disease-Defining T-cell Subsets in the Tumor Microenvironment of Classic Hodgkin Lymphoma

Abstract

Hodgkin lymphoma is characterized by an extensively dominant tumor microenvironment (TME) composed of different types of noncancerous immune cells with rare malignant cells. Characterization of the cellular components and their spatial relationship is crucial to understanding cross-talk and therapeutic targeting in the TME. We performed single-cell RNA sequencing of more than 127,000 cells from 22 Hodgkin lymphoma tissue specimens and 5 reactive lymph nodes, profi ling for the fi rst time the phenotype of the Hodgkin lymphoma-specifi c immune microenvironment at single-cell resolution. Single-cell expression profi ling identifi ed a novel Hodgkin lymphoma-associated subset of T cells with prominent expression of the inhibitory receptor LAG3, and functional analyses established this LAG3 + T-cell population as a mediator of immunosuppression. Multiplexed spatial assessment of immune cells in the microenvironment also revealed increased LAG3 + T cells in the direct vicinity of MHC class II-defi cient tumor cells. Our fi ndings provide novel insights into TME biology and suggest new approaches to immune-checkpoint targeting in Hodgkin lymphoma. SIGNIFICANCE:We provide detailed functional and spatial characteristics of immune cells in classic Hodgkin lymphoma at single-cell resolution. Specifi cally, we identifi ed a regulatory T-cell-like immunosuppressive subset of LAG3 + T cells contributing to the immune-escape phenotype. Our insights aid in the development of novel biomarkers and combination treatment strategies targeting immune checkpoints.

Molecular biology of Hodgkin lymphoma

“…Earlier, largely immunohistochemistrybased analyses of multiple T-cell differentiation markers pointed to a Th2 predominance in cHL [108]. A recent single-cell RNA-seq of cHL tissues also detected a major Th2 subset among Th cells, yet Th1 cells were also enriched in the cHL samples in comparison to reactive lymph nodes [100]. Other studies, mainly based on flow cytometry and CyTOF analysis, however, argued for a Th1 cell predominance in cHL [107,109].…”

“…HRS cells do not only shape their microenvironment by attracting immune cells into their microenvironment, but they also modulate the function and differentiation of cells through secretion of critical factors. For example, HRS cells can induce a Treg phenotype on co-cultured T cells in vitro [100,101]. This is likely mediated by factors secreted by HRS cells and known to induce functional reprogramming of tumor-infiltrating T cells into Th2 cells and Tregs, such as galectin-1 and IL7 [76,93].…”

The evolving landscape of predictive biomarkers in immuno‐oncology with a focus on spatial technologies

“…A novel subtype of T cells with expression of the inhibitory receptor lymphocyte activation gene 3 protein (LAG3) was identified which acted as a mediator of immunosuppression. In the study, increased LAG3 + T cells were shown to be in the direct vicinity of MHC class‐II‐deficient tumor cells 73 . Zhu et al .…”

“…In the study, increased LAG3 + T cells were shown to be in the direct vicinity of MHC class-II-deficient tumor cells. 73 Zhu et al investigated ovarian cancer patients using IMC to identify biomarkers of immunotherapy response. In the study, the authors found that the highest increase in CD8 + T cells and forkhead box protein P3 (FoxP3) + cells was found in patients responding to the combination of durvalumab and tremelimumab.…”

Lymphoma Heterogeneity Unraveled by Single-Cell Transcriptomics

“…Data can be processed with dedicated software, such as Single-Cell_Signature_Explorer and single cell virtual cytometer, the first published method for qualitative and scoring of single gene or gene-set based signatures (68). This method could thus provide informations about: cellular heterogeneity and evolution (our data, in press), clues of cell maturation through pseudo-temporal trajectory analysis (74) or cellular dynamics by RNA velocity determination (70), or definition of GC differentiation state based on CXCR4 and CD86 proteins expression levels, thus confirming sc transcriptomics data (20).…”