Paper Details

ABSTRACT

Introduction: Nodal T follicular helper (T_FH) cell lymphomas (nTFHLs) are characterized by recurrent mutations in epigenetic regulator, TET2 and expression of T_FH biomarkers. Integrated genomic analyses identified co-occurrence of TET2 mutations with TSC2 loss uniquely enriched in nTFHL-NOS, suggesting a novel cooperative genetic mechanism. As TSC2 negatively regulates mTORC1 signaling, concurrent TET2 and TSC2 disruption may cooperatively alter CD4⁺ T cell activation, differentiation, and metabolic programming relevant to T_FH lymphoma biology. However, the biological consequences of combined TET2/TSC2 deficiency remain poorly understood.

Methods: Targeted genomic sequencing data from >200 nTFHL cases were analyzed to define recurrent mutations, copy number alterations and co-occurring genomic events. Primary human CD4? T cells from healthy donors were edited using CRISPR/CAS9 to generate CAS9-transfected control, TET2^-/-, TSC2^-/- and dual knockout (KO) (TET2^-/-;TSC2^-/-) cells. Following anti-CD3/CD28 stimulation, proliferation, apoptosis, activation, memory and exhaustion phenotypes, mitochondrial function, mTOR pathway activity and epigenetic alterations were evaluated. T cell differentiation was assessed under T_H1, T_H2, and T_FH-polarizing conditions. Bulk RNA sequencing was performed to identify differentially expressed genes and enriched pathways.

Results: Genomic analysis demonstrated that TET2 co-occurred with TSC1 (10%) or TSC2 (17.5%) alterations predominantly in nTFHL-NOS and rarely in nTFHL-AI cases. TSC1/2 copy number loss including single-nucleotide variants was identified in nTFHL-AI, while combined genomic alterations occurred in approximately 30% of nTFHL-NOS. Notably, these mutations rarely co-occurred with RHOA^G17V or IDH2^R172 mutations, suggesting an alternative molecular program in nTFHL-NOS subset.

        Under IL-2 and anti-CD3/CD28 stimulation, TET2^-/-;TSC2^-/- cells demonstrated >2-fold greater proliferation than CAS9-transfected cells (p<0.001) and a 1.20-fold increase over TET2^-/- cells (p=0.03), accompanied by reduced apoptosis. Dual KO cells also exhibited increased mitochondrial mass (MitoTracker, p=0.01) and mitochondrial membrane potential (TMRE), consistent with metabolic rewiring. Enhanced mTOR pathway activation was confirmed by increased phosphorylation of S6, S6K, and 4EBP1 proteins. In addition, these cells showed elevated global 5mC levels indicative of epigenetic dysregulation.

       TET2^-/-;TSC2^-/- cells displayed enhanced T cell activation (CD69), memory-associated features (CD45RO, CD62L, IL7-Rα) and reduced exhaustion (TIM-3, TIGIT). Under basal stimulation, the frequency of cells expressing T_FH phenotypes (PD-1?/CXCR5?/ICOS?/BCL6?) increased in TET2^-/-;TSC2^-/- cells (49% vs. 31% in CAS9 and 40% in TET2^-/- cells), while under T_FH-polarizing conditions, this population further increased (55% vs. 38% in CAS9 and 47% in TET2^-/- cells), suggesting enhanced acquisition of T_FH-associated functional characteristics. Differentiation assays further demonstrated reduced alternative T_H1 and T_H2 lineage commitment.

         RNA sequencing revealed upregulation of genes associated with PI3K-mTOR signaling (PIK3R3, PIK3IP1, RPS6KC1, EIF4E3), cellular metabolism (MICOS10, SLC25A20, TOMM40L), T cell activation (KRAS, CD69) and T_FH-differentiation (MAF, PDCD1), with downregulation of exhaustion-associated genes (TIGIT, HAVCR2) in dual KO cells compared with CAS9-transfected cells. Relative to TET2^-/-, TET2^-/-;TSC2^-/- cells further upregulated genes involved in PI3K-mTOR signaling (PIK3R3, RRAGC), mitochondrial metabolism (MICOS10, SLC25A20), and T cell trafficking (S1PR1, CXCR3, CCR5), indicating additional transcriptional remodeling following TSC2 loss. Collectively, these transcriptional changes were consistent with enhanced activation and metabolic phenotype observed experimentally.

Conclusions: Combined TET2 and TSC2 loss was associated with enhanced CD4? T cell activation, proliferation, mitochondrial function, and T_FH-associated functional phenotypes, accompanied by remodeling of PI3K-mTOR signaling and metabolic transcriptional programs. These findings suggest that TSC2 loss modifies the functional and transcriptional state established by TET2 deficiency and identifies candidate pathways relevant to T_FH lymphoma biology. Therefore, our study provides a physiologically relevant human model for dissecting disease mechanisms and prioritizing therapeutic targets in nodal T_FH lymphoma subsets.