Aging-Associated Epigenetic Reprogramming Alters the Germinal Center Reaction and Targets Pathways Related to Lymphomagenesis

Aging is associated with suboptimal germinal center (GC) responses and inferior outcomes of GC-derived lymphomas such as diffuse large B-cell lymphoma (DLBCL). At the molecular level, aging is characterized by epigenetic alterations of DNA CpG methylation and chromatin architecture that ultimately affect cellular functions. The B-cell/T-cell immune synapse during the transitional stages of the GC is governed by a series of epigenetic switches. Frequent mutations in DLBCL directly impact epigenetic regulators and signaling pathways in the immune synapse. Here, we investigated the impact of aging-associated epigenetic alterations in the functionality of the GC response in parallel with biological characteristics of DLBCL in the elderly.

We conducted an integrative characterization of epigenomic, transcriptomic and phenotypic changes of B and T cells during the GC reaction in younger (3-4 months) vs. older (21-23 months) mice by immunophenotyping (flow cytometry), DNA methylation sequencing of sorted sub-populations, single cell (SC) RNA-sequencing (coding and BCR/TCR) and SC-multiomics (integration of RNA-sequencing and ATAC-sequencing). We also performed DNA methylation sequencing, RNA-sequencing and NGS in a cohort of 266 DLBCL including 51 pts >75 y.o.

In agreement with reported phenotypes in elderly humans (Collier D. et al., Nature, 2021), the GC reaction in older mice was characterized by a significative reduction in IgG3+ (p=0.0001) and increased in IgM+ (p=0.009) B cells (FAS+ CD38-); whereas the non-GC B cell compartment (FAS- CD38+) displayed an increase in age-associated B cells (ABCs, p=0.0001) and reduction in follicular B-cells (p=0.004). Furthermore, older mice displayed an expansion of a FAS+CD38+ population of B cells (p=0.0001). Regarding T cells, we observed a global reduction in CD4+ (p=0.01) but not in CD8+ cells; however, older mice showed an expansion of PD1+/CD4+ and PD1+/CD8+ T cells (p=0.0004 and p=0.0003, respectively). Furthermore, older mice displayed increased TFH (p=0.0001), Tregs (p=0001) and ICOS+ Treg (p=0001) populations. SC-RNA-seq of B and T cells validated these alterations and identified transcriptionally-defined sub-populations including expansion of clonal GZMK CD8+ TOX+ T cells (TAA cells) and activation of "cytokine production" in T cells (FDR=3.01e-27), both phenotypes associated with "inflammaging" (Mogilenko et al., Immunity, 2020). Epigenetic changes in older B-cells showed aberrant hypermethylation of gene promoters and hypomethylation of intergenic regions including enhancers, resulting in changes of chromatin architecture and gene expression. In GC B-cells but not naïve B-cells (NBC), genes whose promoter was aberrantly hypermethylated in older mice were enriched for targets of SUZ12 (FDR= 5.1e ^-12), EZH2 (FDR= 5.1e ^-12) and JARID2 (FDR= 6.8e ^-10), key components of the PRC2 complex. Older mice B-cells also displayed decreased chromatin accessibility in genes functionally enriched for "semaphorin-plexin signaling pathway" (FDR=5.3e ^-03) which regulates TFH/B-cell immune synapse as well as decreased expression of linker histone H1 isoforms B-E in GCB cells (q=0.0006; q=0.0003; q=0.008, q=4.64E-10, respectively).

Notably, the age-associated reprogramming observed in the germinal center reaction globally targets pro-tumorigenic pathways that are comparably altered in elderly DLBCL. We observed that older pts (>75 y.o.) vs. younger pts (55-65 y.o.) had increased hypermethylation of gene promoters and hypomethylation of intergenic regions with deregulation of epigenetic modifiers (including PCR2 members) and immune synapse genes (including BCR signaling). There were no differences in the prevalence of specific mutations between these two populations. However, DLBCL in elderly are more frequently of the ABC subtype (~60%, p<0.05) and presented with an inflammatory tumor microenvironment (41% vs 27% in younger DLBCL, p<0.001, corrected by COO) that is characterized by increased infiltration of macrophages (p=0.001), Tregs (p=0.001) and CD8+ PD1 ^highcells (p=0.001), phenocopying changes observed in the germinal center of older mice.

Thus, age-associated epigenetic reprogramming results in perturbation of pathways regulating the B-cell/T-cell immune synapse during the normal germinal center reaction and may underlie some of the biological characteristics observed in elderly DLBCL patients.