Dnmt3a-mutant hematopoietic stem cells generate alloreactive T cells with graft vs host disease and graft vs leukemia effects Free

Clonal hematopoiesis (CH) is an age-related phenomenon caused by expansion of a hematopoietic stem cell (HSC) clone with an acquired leukemogenic mutation. CH is associated with an increased risk of death from cardiovascular disease or hematologic malignancies. Epigenetic regulators account for ~80% of the genes mutated in CH, with DNA Methyltransferase 3a (DNMT3A) mutations being the most common. Clinical studies have shown that CH clones are frequently transmitted from donor to recipient during allogenic stem cell transplantation (up to 27% of patients). DNMT3A mutations remain the most prevalent and patients with donor-derived CH have an increased risk developing graft vs host disease (GVHD) and a decreased risk of leukemia relapse (Frick JCO 2018, Gibson. JCO 2022). These findings may be due to donor-derived DNMT3A mutations within the T cell compartment, as both GVHD and graft vs leukemia (GVL) are T cell mediated, however the effect of CH-associated DNMT3A mutations on GVHD and GVL have not been directly evaluated.

To determine whether mutations in DNMT3A affect T cell function leading to GVHD and GVL, we used a conditional knockin murine model of a common CH-associated DNMT3A point mutation, Dnmt3a^R878H/+(Loberg. Leukemia 2019). We found that after ~ 24 weeks of mutant allele induction in HSCs, Dnmt3a^R878H/+mice developed a severe terminal ulcerative dermatitis marked by epidermal hyperplasia and an inflammatory lymphocytic infiltrate. Single cell suspensions prepared from the skin of wild type (WT) and mutant mice were notable for an increase in conventional CD4+ T cells (p< .04) and CD4+ effector memory T cells (TEM, p<.04) in Dnmt3a^R878H/+mice. Dnmt3a^R878H/+mice also had elevated serum inflammatory cytokines and chemokines compared to WT including IL-6, CCL5, and MCP-1 (p<.05). Immunophenotyping of splenocytes revealed similar percentages of conventional CD4+ and CD8+ T cells in WT and mutant mice but significantly increased CD4+ TEM cells (p<.001), CD8+ central memory T cells (p<.02), and decreased naïve CD4+ (p<.0001) and naïve CD8+ (p<.001) T cells in Dnmt3a^R878H/+mice. Methylation and transcriptome profiling were performed on sorted conventional CD4+ and CD8+ T cells to determine whether this activated T cell phenotype was due to Dnmt3a-dependent changes in DNA methylation and gene expression. We identified significant promoter and gene body hypomethylation and transcriptional upregulation of genes in pathways involved in lymphocyte differentiation, activation, and inflammation CD4+ and CD8 + Dnmt3a^R878H/+T cells compared to WT.

To functionally test whether naïve T cells from Dnmt3a^R878H/+mice had increased alloreactivity, we adoptively transferred naïve CD4+ or CD8+ T cells into NSG mice. Dnmt3a^R878H/+naïve CD4+ T cell recipients had earlier clinical signs of GVHD (ruffled fur, hunched posture) than WT naïve CD4+ T cell recipients (7 vs 21 days). Dnmt3a^R878H/+naïve CD4+ T cell recipients also had a more robust expansion of CD4+ T cells in the spleen by flow cytometry (52% vs 31% live cells, p<.01) and more severe skin, liver, and colon GVHD by histopathology scoring. Although Dnmt3a^R878H/+naïve CD8+ T cell recipients developed clinical signs of GVHD earlier ( 21 vs > 42 days) and had more severe skin, liver, and colon GVHD by histopathology scoring than WT naïve CD8+ T cell recipients, GVHD onset and severity was worst in Dnmt3a^R878H/+naïve CD4+ T cell recipients. We then directly evaluated the GVL activity of Dnmt3a^R878H/+and WT T cells using an aggressive AML cell line, C1498. In vitro, Dnmt3a^R878H/+T cells killed more C1498 cells at lower effector to tumor ratios (1:1, 1:2) than WT T cells (p<.04). In vivo, WT mice had a median survival of 26 days whereas > 50% Dnmt3a^R878H/+mice survived the C1498 challenge (p=0.009). Although there were no differences in CD4+ splenocytes isolated from WT and mutant mice 3 weeks after C1498 challenge, Dnmt3a^R878H/+mice had more TEM and GrzB+/CD95+ CD8+ splenocytes. All surviving Dnmt3a^R878H/+mice survived rechallenge with the same dose of C1498 cells whereas control WT mice succumbed by week 5. Collectively, we demonstrate that HSCs with CH-associated DNMT3A mutations can give rise to hyperactivated T cells with the potential to drive GVHD, which appears to be predominately mediated by conventional CD4+ T cells, as well as GVL mediated by CD8+ T cells, in allogeneic stem cell transplant recipients.