Abstract
Hematopoietic cell transplantation (HCT) is a curative therapy for the treatment of malignant and non-malignant bone marrow diseases. The major complication of this treatment is a highly inflammatory reaction called Graft-versus-Host Disease (GvHD). Here, transplanted donor T cells cause massive tissue destruction and inflammation in the main target organs liver, skin and the intestine. Currently, this inflammatory reaction can be treated successfully using strong immunosuppressive agents. One efficient group of immunosuppressants are calcineurin inhibitors such as Cyclosporin A (CsA) and Tacrolimus (FK506). These treatment strategies target all T lymphocytes subsets equally and do not separate GvH from the desirable Graft-versus-Leukemia (GvL) effect. Therefore, we aimed to find immunological targets on alloreactive T cells in order to develop novel treatment strategies, which selectively modulates alloreactive T cells without impairing the GvL effect or hematopoietic immune reconstitution. The aim of this thesis was to develop a predictive marker panel to track alloreactive T cells in the peripheral blood (PB) of murine allo-HCT recipients. In clinically relevant model of aGvHD we demonstrated that alloreactive T cells have a distinct surface marker expression profile and can be detected in the PB before aGvHD manifestation. Based on our data, we propose a combinatory panel consisting of 4 surface markers (a4b7 integrin, CD162E, CD162P und CD62L) on circulating CD8+ T cells to identify the risk of aGvHD after allo-HCT. Since tumor necrosis factor receptor superfamily (TNFR SF) members are involved in several immunological processes, we did extensive surface marker expression analysis of several TNFR superfamily members and other immunomodulatory molecules on conventional and regulatory T cells (Tcons vs. Tregs) on different time points during aGvHD progression. The aim of this study was to find subset-specific immunomodulatory molecules on recently activated Tcons and Tregs. We found that GITR, 4-1BB and CD27 were highly expressed on alloreactive and naive Tregs. In contrast, PD1 expression was highly upregulated on recently activated alloreactive Tcons. The data of this study serves as basis for future approaches, which aim to develop T cell subset specific therapeutic antibody fusion proteins. a4b7 integrin and CD162P (P-Selectin ligand) are highly upregulated on alloreactive T cells and mediate the infiltration of these cells into GvHD target organs. We developed recombinant (antibody) fusion proteins to target these two homing molecules and could show that antibody-based fusion proteins are superior to ligand-based fusion proteins regarding production efficiency and binding affinity. Therefore, we propose for future studies to focus on the described antibody-based fusion proteins for the selective targeting of T cells. Since the widely used calcineurin inhibitors are impairing the desirable GvL effect, we investigated if selective NFATc1 inhibition might be a novel strategy to prevent or reduce alloreactivity, while hopefully maintaining the GvL effect. In particular, we addressed the role of the isoform NFATc1 and inhibited its posttranslational modification by SUMO (Small Ubiquitin-related Modifier). Indeed, inhibition of NFATc1 SUMOylation resulted in reduced inflammation and increased Treg frequencies in a murine MHC major mismatch aGvHD model. Conclusively, we showed that alloreactive T cells can be identified by their surface profile in the PB of allo-HCT recipients before aGvHD symptoms appeared. Furthermore, we introduced a approach to selectively target alloreactive T cells by antibody fusion proteins, which might serve as a novel strategy to separate GvH from GvL. Additionally, we demonstrated that averted posttranslational modification of NFATc1 by SUMOylation serves as potential target to reduce alloreactivity of T cells.
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