Germinal Center Program De-Synchronization and Intra-Patient Heterogeneity in Follicular Lymphoma B-Cells Revealed By Integrative Single-Cell Analysis

Introduction

Follicular lymphoma (FL) is an indolent B-cell neoplasm with histological, phenotypical and transcriptional features of germinal center (GC) B-cells, suggesting that FL B-cells are "frozen" in a GC B-cell state. In normal GC B-cells, affinity maturation relies on iterative cycles of somatic hypermutation and proliferation followed by affinity-based selection. Thus GC B-cells are functionally heterogeneous. Although FL sub-clonal genetic diversity is well documented, tumor cell functional heterogeneity in FL has not been studied in detail. Bulk-level transcriptomic analyses fail to capture cellular heterogeneity and are thus insufficient to compare FL B-cells to the heterogeneous GC B-cell subset. Here, we used integrative single-cell analysis of phenotype, gene expression and IGH sequence to assess and compare functional heterogeneity in FL B-cells and GC B-cells.

Methods

Normal B-cells were sorted from spleens or tonsil of healthy human donors (n=4), and FL B-cells from diagnostic lymph node biopsies (n=5), all available as frozen viable cell suspensions. Single normal or malignant B-cells were sorted by flow cytometry for parallel analysis of phenotype (9-color panel), gene expression (91-gene panel by microfluidic RT-qPCR), and IGH sequence (RT-PCR followed by Sanger sequencing; FL B-cells only). Custom and existing bioinformatics analysis pipelines were combined for quality control and cell filtering, dimensionality reduction (PCA, t-SNE), clustering, pseudo-time analysis, IGH sequence analysis and integrative data analysis.

Results

In normal B-cells, single-cell gene expression profiling segregated major cell types (antibody-producing cells, memory B-cells and GC B-cells) and enabled the reconstruction of differentiation trajectories within cell types. Specifically, our data revealed a cyclic continuum of transitional GC B-cell states where synchronized expression of gene modules characterized human GC B-cell functional heterogeneity. Single-cell gene expression profiles defined FL B-cells as an independent cell type, distinct from GC B-cells. In particular, the expression of most characteristic GC-associated genes was de-synchronized in FL B-cells from all patients. Clustering of single FL B-cell 91-gene expression profiles revealed inter-patient and intra-patient heterogeneity, and defined common FL B-cell subsets found in most patients. Intra-patient heterogeneity was not linked to sub-clonal divergence as defined by IGH sequencing.

Conclusions

Using integrative single-cell analyses, we have shown that synchronized expression of characteristic gene modules drives GC B-cells through a cyclic continuum of transitional states. De-synchronization of that program in FL B-cells, and substantial intra-patient gene expression heterogeneity, suggest that FL B-cells are functionally plastic and not blocked to a GC B-cell state.