Desynchronization of the Germinal Center Dynamics and Remodeling of the Tumor Microenvironment Characterize KMT2D-Driven Lymphomagenesis
Authors: Brisou Gabriel , Zala Manon , Gil Laurine , Pagano Giulia , Bru Agnès , Cervera-Marzal Inaki , Potier Delphine , Nin Sebastien , Chasson Lionel , Laprie Caroline , Huet Sarah , Salles Gilles , Milpied Pierre , Nadel Bertrand , Roulland Sandrine 
Published: October 2019

Abstract
Follicular lymphoma (FL) is a prototypical example of germinal center (GC) derived B-cell lymphoma. Using a mouse model recapitulating the sporadic occurrence of the FL hallmark BCL2/IGH translocation in healthy individuals, our previous work demonstrated that FL genesis is a dynamic process that requires multiple re-entries of BCL2+ memory B-cells into the GC to ultimately accumulate in lymphoid organs. In line with this, using single-cell gene expression analysis of human FL vs normal GC B-cells, we recently discovered that FL cells are not 'frozen' at a particular GC maturation stage but instead exhibit a major desynchronization of the GC-specific expression program (Milpied et al. Nat Immunol in press). Since KMT2D loss-of-function mutations and BCL2 translocations are the 2 main alterations in FL, we hypothesized these 2 genetic events might explain the GC program desynchronization we observe in humans.
To explore the in vivo consequences of Kmt2d inactivation with Bcl2 overexpression in regulating GC/memory dynamics, we transduced bone marrow progenitors carrying B-cell-specific conditional Cd19-Cre Kmt2dflox/flox alleles with a retrovirus encoding human BCL2 or reporter alone, followed by iv transplantation into lethally irradiated WT recipients. Only double-mutant Kmt2d-/-Bcl2+ mice manifested with GC-derived lymphomas in chronically challenged animals, recapitulating histological and phenotypic features associated with human FL progression from early preneoplastic lesions to overt FL-like tumors. We used integrative single-cell analysis of surface phenotype (10-color panel), gene expression (88-gene panel by microfluidic RT-qPCR) and IGH clonality to deconvolute cellular heterogeneity of flow-sorted GC and memory B-cells after acute (day 10) or chronic T-cell dependent immunization in double-mutant vs. single Bcl2+, Kmt2d-/- or WT mice, retaining >4000 cells for downstream analysis. Populations of WT GC B-cells were molecularly heterogeneous and spanned a cyclic continuum of transitional B-cell states polarized between the light and dark zone where synchronized expression of gene modules characterized mouse GC functional identity. In acute responses, single and double-mutant mice formed GC similar to control chimera mice, and single GC B-cells from all genotypes clustered together with WT GCs, suggesting unperturbed GC transcriptional dynamics upon first antigen encounter. However, preneoplastic/tumoral Kmt2d-/-Bcl2+ mice after chronic challenge showed massive GC hyperplasia and single B-cells expressed a distinct transcriptional signature that clustered separately from WT GC or memory cells. Specifically, murine lymphoma B-cells sorted with a GC-like phenotype accumulated in transitional cell states where the synchrony of most GC-specific co-expression patterns was progressively lost whereas expression of cytokines (Lta) or surface markers linked to GC to memory transition (Gpr183, Cxcr3) became markedly expressed, indicating that Kmt2d-deficient lymphomas were not blocked at a particular GC stage.
Given the importance of T-cell help for the fate 'decisions' of GC-to-memory B-cells, we further explored whether tumor cell-intrinsic factors may affect immune cell phenotypes thereby supporting the GC gene expression desynchronization. Using flow cytometry, we found that Kmt2d inactivation instructed a progressive remodeling of the tumor microenvironment with an increased recruitment of CD4+ T-follicular helper (TFH) (n=21, p<0.01). Using droplet-based single-cell RNA-seq to profile total spleens from 2 WT mice and 2 lymphomas (>9000 cells), we revealed a TFH cluster with an activation signature distinct from normal TFH and a concomitant expansion of exhausted CD8+ T-cells strongly co-expressing inhibitory receptors (Lag3, Tim3, Pdcd1), thereby indicating that Kmt2d inactivation in B-cells may favor lymphoma formation by shaping the FL tumor supportive niche and contributing to immune evasion.
In summary, our integrative single-cell analyses in murine lymphomas revealed that Kmt2d mutations in FL not only instruct B-cell intrinsic effects involving the desynchronization of the normal GC expression program, but also trigger a concomitant re-education of a tumor-supportive immune microenvironment, establishing for the first time a key link between the most frequent epigenetic alteration and the FL microenvironment.

Disclosures
Salles: F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Celgene: Honoraria, Other: Advisory Board, Research Funding; Janssen: Honoraria, Other: Advisory Board; Novartis: Consultancy, Honoraria; Takeda: Honoraria; Servier: Honoraria, Other: Advisory Board; Epizyme: Honoraria; Merck: Honoraria; BMS: Honoraria, Other: Advisory Board; Morphosys: Honoraria; Acerta: Honoraria; Amgen: Honoraria; Gilead: Honoraria, Other: Advisory Board; Pfizer: Honoraria; Servier: Honoraria; Abbvie: Honoraria.