Identification of Somatic Mutations in Expanded T Cell Clones of Patients with Chronic Graft-Versus-Host Disease (GVHD)

Background: GVHD is the main complication in allogeneic hematopoietic stem cell transplantation (allo-HSCT). Chronic GVHD (cGVHD) develops in 60-80 % of allo-HSCT survivors causing marked morbidity and mortality. Donor alloreactive T cells are the key drivers in the development of cGVHD, since they recognize and damage the host target tissues by cytokine release and direct cytolysis. Unbalanced reconstitution of T cell pool has been suggested to be a crucial factor in the development of cGVHD. In particular, disease-associated T cell receptor (TCR) clones have been detected and abnormally expanded within the blood and affected tissues in cGVHD patients. It is unclear why these T cell clones do not undergo physiologic FAS-mediated apoptosis. Here, we hypothesized that acquired somatic mutations in T cells may lead to skewed clonal expansions and contribute to the pathogenesis of cGVHD.

Methods and Results: We collected blood samples from patients with cGVHD (n=39) and healthy controls (n=11). CD4+ and CD8+T cells were sorted with magnetic beads, and genomic DNA was extracted. TCR repertoire was analyzed by T cell receptor b-chain (TCRB CDR3) deep sequencing. Somatic mutations in CD4+ and CD8+ T cells were discovered using a custom NGS gene panel consisting of 2000 genes that are essential for innate and adaptive immunity.

Interestingly, the index cGVHD patient had a marked clonal expansion within the CD4+T cell pool, and TCRB deep sequencing revealed one specific TCRVB clone (30-01, 50% of CD4+T cells). By performing the immune gene panel sequencing, we discovered 3 somatic mutations in mTOR, TLR2 , and NFkB2 genes in the expanded CD4+T cell clone. The mutations persisted during a 2-year follow-up and were not detected within the CD8+T cell compartment. To verify the sequencing results, targeted mTOR, TLR2, and NFkB2 sequencing was performed with an in-house developed deep (1,000x coverage) amplicon sequencing panel. Subsequently, 128 cGVHD patients were screened to assess whether the discovered mutations were recurrent. In total, the mTOR mutation was discovered in 3 patients, the TLR2 mutation in 19 patients, and the NFkB2 mutation only in the index case. Strikingly, the mTOR mutation was also identified in the sclerodermatic skin lesion from the index patient with a low variant allele frequency (VAF), and immunofluorescence staining confirmed CD4+T cell infiltration in the skin. In T cells of the index patient's sibling donor, no mTOR, TLR2, or NFkB2 mutations were detected. In a cohort of 29 additional healthy individuals, no mTOR or NFKB2 mutations were detected in sorted T-cell fractions, but small VAF TLR2 mutations were found in 7 controls implying them not to be GVHD related.

To study the functional consequences of the identified mutations, we constructed expression vectors containing the mutations for transfection of HEK-293 cells. Alterations in transcriptional regulation were evaluated by analyzing mRNA expression levels of a subset of mTOR, TLR2 and NFkB2 downstream targets byqRT-PCR. The levels of MAPKAP1, RPS6, RHOA, NFKB1, NFKBIL1, NFRKB, ELK1, FOS, MAP3K1, MAP3K7 , and MAP4K4 were significantly increased in mutant cells compared to wild-type controls.

Finally, the NFkB2 mutation was located in the c-terminal domain which is known to play an important role in the ubiquitination and partial proteolysis from NFkB2 (p100) to NFkB2 (p52). To analyze the molecular balance between p100 and p52, immunoblotting was pursued and revealed that the mutation induced overexpression of p52.

Conclusions: We identified novel somatic mutations in T cells of patients with cGVHD. In our index patient, the mutations were discovered in the aberrantly expanded CD4+T cell clone. As the mutations were not observed in the T-cells of the sibling donor, we hypothesize that they were acquired during the immune activation and rapid expansion of T cells after the allo-HSCT. The discovered mTOR mutation was found to be recurrent in 3 patients based on a screening cohort of 128 cGVHD patients, but was not detected in healthy individuals. Functional analyses suggested the mutations to be gain-of-function of nature, and thus inducing marked activation of the mTOR and NFkB2 signaling pathways. As both pathways are crucial in T cell activation and function, our data may provide a novel explanation for the aberrant, persistent T cell activation in cGVHD and pave the way for novel targeted therapies.