Paper Details

ABSTRACT

Introduction: Loss of Tet1 expression in a murine model drives late-onset follicular lymphoma (FL), and Tet1 promoter hypermethylation and downregulated expression have been observed in B cells from FL patients. Ninety percent of FL patients carry the t(14;18) chromosomal rearrangement, which increases BCL2 expression, and Bcl2 transgenic mice also develop FL after a long latency period. Given that both alterations independently predispose patients to FL, we modeled whether combining Tet1 deficiency with Bcl2 overexpression in a murine model would accelerate lymphomagenesis. Instead, we report that Tet1 deficiency, alone or in combination with Bcl2 overexpression, drives an increased propensity for plasma cell differentiation and autoimmune-like polyclonal gammopathy.

Methods: Vav promoter-driven Bcl2-overexpressing transgenic mice were crossed with Vav promoter tet-off transactivator (Vav-tTA) mice to allow doxycycline-regulated shRNA expression targeting Tet1 or Tet2, with Renilla luciferase used as a control. Vav-Cre Tet1 floxed mice were also generated without Bcl2 overexpression and crossed with Prdm1-YFP reporter mice to track plasma cell differentiation. B-cell development was characterized using flow cytometry to quantify developing B cells, germinal center B cells, and plasma cell populations. Serum immunoglobulin (Ig) measurements and immunohistochemistry were performed in cohorts of aging mice that were monitored for overall survival. B-cell receptor (BCR) signaling strength was measured by Ca²? flux assays, and kinase activation was assessed by phospho-flow cytometry. Plasma cell formation and class-switch recombination were measured by ex vivo stimulation with LPS, anti-CD40, IL-4, and IL-21. Proliferation was assessed using CellTrace, and apoptosis was measured by Annexin V staining. Plasma cell assays were also performed using a pharmacological inhibitor of TET enzymes (Bobcat-339). Mice were immunized with NP-KLH to measure germinal center B-cell and plasma cell frequencies by flow cytometry and serum immunoglobulin levels by NP-specific ELISA in the context of Tet1 knockdown versus restoration following doxycycline treatment in vivo. We further analyzed the molecular changes in Tet1-deficient naïve B cells and plasma cells by performing transcriptome profiling and base-resolution hydroxymethylation mapping to define the epigenetic changes caused by Tet1 loss in naïve B cells and plasma cells. ChIP-PCR for Tet1 was also performed to quantify chromatin occupancy at plasma cell gene loci (e.g., Prdm1).

Results: Tet1 loss in Bcl2-overexpressing B cells resulted in a rapidly fatal autoimmune-like polyclonal gammopathy driven by an increased propensity to generate antibody-secreting plasma cells. We show that Tet1 expression increases as naïve B cells differentiate into plasma cells and that its loss enhances both Ca²? flux and kinase activation following BCR stimulation of naïve B cells, while also increasing the frequency of class switching. In vivo immunization similarly promoted enhanced germinal center and plasma cell formation, along with an elevated serum Ig response, in Tet1-deficient mice. Hydroxymethylation and transcriptomic changes reflected an underlying epigenetic priming of naïve B cells toward a state of increased activation potential. This was consistent with epigenomic changes at the Prdm1 locus and its upregulated expression in differentiating Tet1-deficient plasma cells. Restoring Tet1 activity by switching off RNAi-mediated knockdown through doxycycline treatment reversed the excessive antibody production and aberrant plasma cell phenotypes both in vitro and in vivo.

Conclusion: Our study identifies a previously unrecognized role for Tet1 in the regulation of plasma cell differentiation. Importantly, in the context of Bcl2 overexpression, Tet1 loss drives a fully penetrant autoimmune-like disease associated with excessive Ig levels. The reversibility of this phenotype following Tet1 restoration suggests that Tet1 may represent a potential therapeutic target for plasma cell diseases.