Abstract
Allogenic hematopoietic cell transplantation (allo-HCT) is an established therapy for the treatment of malignant diseases such as leukemia or lymphoma. Unfortunately, this often leads to an immunological complication, termed graft-versus-host disease (GvHD), in which donor T cells attack host tissues. Patients with acute GvHD can be efficiently treated with immunosuppressive agents such as cyclosporin A and tacrolimus. These agents inhibit the phosphatase calcineurin, which leads to suppression of nuclear factor of activated T-cells (NFAT). However, inhibition of calcineurin causes severe side effects and impairs the graft-versus-leukemia (GvL) effect. Therefore, we evaluate new therapeutic options.
Previously, we have demonstrated that posttranslational modification of NFATc1 by SUMO (Small Ubiquitin-like MOdifier) modulates its transcriptional activity in vitro (Nayak et al. 2009. J Biol Chem 284:10935-46). To elucidate the importance of NFATc1 SUMOylation in vivo, we generated an NFAT mutant mouse with lysine to arginine exchanges within the C-terminal SUMOylation motifs, Nfatc1K702/914R, encoding NFATc1ΔSumo. NFATc1ΔSumo mice were healthy and developed a normal lymphoid compartment. In line with our former in vitro studies, however, NFATc1ΔSumo CD4+ T cells produced more IL-2 and less effector lymphokines like IFN-γ when challenged ex vivo. Since enhanced IL-2 levels can protect from GvHD, we compared NFATc1ΔSumovs WT T cells in an murine MHC major mismatch allo-HSCT model (C57BL/6, H-2b into BALB/c, H-2d), leading to acute GvHD. For noninvasive bioluminescence imaging of transplanted T cells, we crossed NFATc1ΔSumo mice with firefly luciferase-expressing mice. Recipients of NFATc1ΔSumo T cells survived much longer than WT T-cell recipients, correlating with a significant reduction of in vivo expansion and GvHD target organ infiltration. Surface expression of α4β7-integrin, which guides T cells into the intestine, was slightly decreased on CD4+ T cells of NFATc1ΔSumo mice. Accordingly, immunofluorescence microscopy revealed reduced NFATc1 SUMOylation-deficient CD4+ T cells infiltrating the gastrointestinal tract. Importantly, intracellular TNF-α and IFN-γ levels were significantly decreased in alloreactive NFATc1ΔSumoT cells.
In contrast, CD4+ CD25+ Foxp3+ regulatory T (Treg) cells increased in mice with transplanted NFATc1ΔSumo T cells. To evaluate whether higher IL-2 production from conventional T cells (Tcons) would enhance Treg frequency, we transplanted NFATc1ΔSumo or WT Tcons, always in combination with WT Tregs to suppress GvHD in vivo. Indeed, WT Tregs frequencies were 2-fold higher in the presence of Tcons from NFATc1ΔSumo mice as compared to WT Tcons. Consequently, expansion of NFATc1ΔSumo alloreactive Tcons was inhibited. Accordingly, an in vitro suppression assay demonstrated that NFATc1ΔSumo regulatory T cells (Tregs) exhibit similar suppressive capacities as WT Tregs and, thus, may mainly benefit from the beneficial condition provided by NFAT1ΔSumoTcons.
Conclusively, NFATc1 SUMOylation in T cells is critical for balancing inflammation and tolerance by regulating the ratio of Tcons vs Tregs. We postulate that averted NFATc1 SUMOylation ameliorates inflammatory diseases due to higher IL-2 production, which supports Treg proliferation. Blocking NFATc1 SUMOylation in T cells before allo-HSCT poses a potential therapeutic option similar to IL-2 treatment against GvHD.
Disclosures
No relevant conflicts of interest to declare.
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