Tet2 Loss and IDH2^R172K Mutation Develop T_FH-Cell-like Lymphoma with Restricted T_H1 Differentiation Program and Cognate Interaction with B Cells

Introduction:

Nodal lymphomas of T-follicular helper cells (n-TFHLs) are considered neoplasms of mature T_FH cells and are classified by the World Health Organization (WHO) under one umbrella with three subtypes: angioimmunoblastic-type (n-TFHL-AI), nodal TFHL, follicular type (n-TFHL-F), and nodal TFHL, not otherwise specified (n-TFHL-NOS). TET2 mutations in n-TFHL-AI subtype frequently co-occur with IDH2^R172 mutations.

Methods:

To elucidate the cooperative role of TET2 and IDH2^R172K mutation in n-TFHL-AI pathogenesis, we generated a murine model with Tet2 loss and IDH2^R172K knock-in mutation in CD4+ T cells (Tet2^-/-/IDH2^R172K) by crossing Tet2^Flox/Flox/IDH2^R172K mice with Cd4-Cre transgenic mice. This resulted in Tet2 exon 3 deletion and IDH2^R172K knock-in mutation specifically in CD4+ T cells. Murine tumors were comprehensively evaluated through immunohistochemistry and flow cytometry. Transcriptomic (RNA-seq), 5-hydroxymethylcytosine (5hmC), and 5-methylcytosine (5mC) profiling, and genomic (whole exome sequencing [WES]) analyses were conducted on both earlier time points (preneoplastic) and neoplastic cells. Functional validation involved proliferation, T-cell polarization, and Western blotting assays.

Results:

The transcriptomic analysis (p < 0.05) in n-TFHL-AI (double-mutant) cases showed enrichment of T_FH-like signature, IL-12-signaling, NF-ΚB, and downregulation of T_H1 cell differentiation (including IFNγ signaling). Evaluation of double-mutant mice revealed an earlier tumor onset than Tet2 mutant mice, with a median survival of 1.3 years compared to 1.7 years (p=0.006), respectively. With chronic stimulation by T cell-dependent antigens (sheep red blood cells [SRBCs]), these mice showed B-cell expansion with a 100% incidence of lymphoid neoplasms, in contrast to PBS-treated mice, where myeloid and lymphoid diseases occurred at an equal ratio. Genomic analysis of murine tumors revealed a unique mutation spectrum in metabolic genes (Ata3d3a, Atp1a3, Ppp4r3a, and Nemf), T- and B-cell interaction (Gnai3), Stat pathway (Jak1), and DNA repair (Zfp451). Transcriptomic analysis of tumors showed downregulation of Tbx21, increased T_FH-related genes, and increased expression of Bcl6 with downregulation of its known suppressed genes.

Transcriptomic analysis of murine CD4+ T cells isolated at 12 weeks revealed downregulation of genes associated with T_H1-cell differentiation and Trp53 signaling, concurrent activation of T-cell receptor (TCR) and MAPK signaling, and API-complex. We demonstrated elevated levels of 2-hydroxyglutarate (2-HG) in double-mutant cells, and enasidenib (IDH2^R172 inhibitor) treatment significantly decreased 2-HG levels and increased Tbx21 expression. Mutant cells showed a proliferative advantage in IL-2 and IL-21 in vitro conditions, resulting in enhanced TCR, AKT, ERK1/2, p-65, and inactivation of p-FOXO1 protein. While these cells showed a growth advantage in in vitro culturing with T_FH conditions, their growth and polarization were impaired in T_H1 conditions, indicating a differentiation block.

Tet2^-/-/IDH2^R172K mice (12-week) with SRBCs treatment revealed a significant induction of B and CD4+ T- cell populations compared to wild-type (WT) mice (p=0.01). In vitro co-culturing experiments demonstrated that Tet2^-/-/IDH2^R172K mutant CD4+ T-cells confer a substantial proliferative advantage to B-cells (p=0.021). Notably, these double-mutant CD4+ T cells exhibited superior proliferation compared to WT CD4+ T-cells when co-cultured with B-cells isolated from WT or double mutant mice (p=0.004). When Tet2^-/-/IDH2^R172K neoplastic cells with whole tumor were allografted into NOD scid gamma (NSG) mice, lymphoma developed within three months, but also showed numerous B-cells in the TME. This suggests that the propagation of neoplastic T-cells likely require B cell help. Overall, the T_FH program remained intact in transplanted cells, and a global decrease in 5hmC levels and an increase in 5mC levels were observed.

Conclusion:

Tet2^-/-/IDH2^R172K murine CD4+ T cells exhibited hyperactive TCR signaling, impaired DNA damage response, and NF-ΚB activation as key oncogenic transitions. Both in vitro co-culturing and the allografting of neoplastic Tet2^-/-/IDH2^R172K cells showed dependence on B-cells, suggesting that interrupting T- and B-cell interaction could be a therapeutic approach.

Feldman:Seattle Genetics: Research Funding; Zeno Pharmaceuticals: Patents & Royalties.