Averted NFATc1 Sumoylation in Alloreactive T Cells Ameliorates Acute GvHD

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, Nfatc1^K702/914R, encoding NFATc1^ΔSumo. NFATc1^ΔSumo mice were healthy and developed a normal lymphoid compartment. In line with our former in vitro studies, however, NFATc1^ΔSumo 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 NFATc1^ΔSumovs WT T cells in an murine MHC major mismatch allo-HSCT model (C57BL/6, H-2^b into BALB/c, H-2^d), leading to acute GvHD. For noninvasive bioluminescence imaging of transplanted T cells, we crossed NFATc1^ΔSumo mice with firefly luciferase-expressing mice. Recipients of NFATc1^ΔSumo 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 NFATc1^ΔSumo 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 NFATc1^ΔSumoT cells.

In contrast, CD4⁺ CD25⁺ Foxp3⁺ regulatory T (Treg) cells increased in mice with transplanted NFATc1^ΔSumo T cells. To evaluate whether higher IL-2 production from conventional T cells (Tcons) would enhance Treg frequency, we transplanted NFATc1^ΔSumo 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 NFATc1^ΔSumo mice as compared to WT Tcons. Consequently, expansion of NFATc1^ΔSumo alloreactive Tcons was inhibited. Accordingly, an in vitro suppression assay demonstrated that NFATc1^ΔSumo regulatory T cells (Tregs) exhibit similar suppressive capacities as WT Tregs and, thus, may mainly benefit from the beneficial condition provided by NFAT1^ΔSumoTcons.

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.