Graft-versus-host disease (GVHD) is a major complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT), contributing to high levels of morbidity and mortality. Calcineurin inhibitors (CNIs), including cyclosporine and tacrolimus, are commonly administered in clinical settings to prevent GVHD but have substantial negative side effects such an elevated risk of infections. The regulation of the Nuclear Factor of Activated T cells (NFAT), which is essential for T cell activation and function, is disrupted by CNIs. Our previous studies have shown that the use of NFAT-deficient T cells in allo-HSCT reduces GVHD while preserving the beneficial graft-versus-leukemia (GVL) response. In this context, we explored the impact of murine cytomegalovirus (MCMV) infection on the progression of GVHD and investigated whether T cell-specific NFAT ablation retains its protective effect. Our findings show that while acute MCMV infection worsened clinical outcomes in allotransplanted mice, the presence of NFAT-deficient T cells still conferred a protective effect against GVHD when compared to wild-type (WT) T cells. Even in the clinically challenging scenario of transplantation from uninfected donors into MCMV seropositive recipients, allo-HSCT with NFAT-ablated T cells maintained lower clinical scores. Moreover, NFAT-ablated CD8+ T cells exhibited expanded MCMV-specific inflated memory and effectively controlled infection. To explore a more clinically relevant scenario, where human donor T cells are typically not immunologically naïve, donor cells were pre-stimulated in vitro with anti-CD3/CD28 or in vivo with MCMV prior to transplantation. In contrast to our earlier observations with naïve T cells, mice receiving pre-activated NFATc1-deficient T cells, displayed significantly reduced survival. To investigate the underlying mechanisms, we employed single-cell RNA sequencing (scRNA-Seq) to analyze liver lymphocytes 14 days post-transplantation. This analysis revealed profound changes in gene expression in pre-activated NFATc1-deficient T cells, including reduced expression of genes associated with regulatory T cell induction and maintenance, as well as genes promoting T cell exhaustion. Conversely, pre-activated NFATc1-deficient T cells upregulated genes involved in pro-inflammatory cytokine production, and cellular stress responses. These findings indicate that, while transplantation of naïve NFAT-deficient T cells offers protection against GVHD and enhances survival, regardless of acute or latent MCMV infection, the absence of NFATc1 in pre-activated donor T cells disrupts immune regulatory mechanisms, exacerbating GVHD and increasing mortality. This underscores the necessity of using naïve T cells when considering NFATc1 ablation as a therapeutic strategy for GVHD prevention.
Nadine Hundhausen (Mon,) studied this question.