Our understanding in GvHD biology has advanced substantially; yet, the pathophysiology of gut GvHD is poorly understood partially due to the lack of gut biopsies for research and the difficulties to directly translate data from murine experiments into humans. The aim of this dissertation was therefore to analyze immune cell infiltrates and biomarkers in gut biopsies of patients after allogenic stem cell transplantation and to explain some of the newer findings on the role of the microbiome by analyzing bacterial metabolites in vitro .
CD4+ T cells and CD8+ T cells were analyzed by means of single antibody immunohistochemistry (IHC). CD4+ T cells were found to be correlated with CD8+ T cell infiltration and epithelial apoptosis whereas CD8+ T cells were upregulated in GvHD and correlated with crypt loss and epithelial apoptosis confirming the primacy of T cell infiltrates in development and exacerbation of GvHD.
Foxp3+ and IDO+ cells were analyzed by IHC in gut biopsies of ASCT patients and the respective mRNA expression was analyzed by qPCR. Both Foxp3+ cells and IDO+ cells significantly upregulated at protein and mRNA level with exacerbation of GvHD, and, Foxp3 and IDO expression strongly correlated to each other suggesting an immunoregulatory loop reactively activated to dampen the T cell response. Using double immunofluorescence, existence of both, the expected CD4+Foxp3+ and the new population of CD8+ Foxp3+ Tregs were discovered which may be crucial for GvHD resolution.
IL-17+ cells were analyzed by IHC and immunofluorescence in colon biopsies of ASCT patients which were significantly downregulated in GvHD. In line with protein expression, the transcription factor RORC significantly downregulated in GvHD. Double immunofluorescence depicted these cells are non T cells but co-express CD117 (c-kit) confirming the innate origin of IL-17+ cells, more likely to be type 3 innate lymphoid cells. This suggests that IL17+ cells beyond TH17 cells exist also in humans which may be protective in GvHD by production of protective IL22.
First phase of gene profiling revealed four possible candidate genes: CYP27A1, CYP27B1, VDR and PXR that were highly regulated in GvHD and two genes: VDR and CYP27A1 could be a future biomarker. The predominance of Vitamin D dependent genes as significant biomarkers in GI tissue further supports the central role of immunoregulation in GvHD related inflammation.
As many of these regulatory loops are activated by the interplay between intestinal microbiota and immune cells, we revealed the importance of bacterial metabolite indoxyl sulfate as a potential immunomodulator that inhibited maturation and activation of human monocyte-derived dendritic cells in vitro.
In summary, our data underline that GvHD is a result of an imbalance between alloreactive T cells and protective regulatory cells and ILCs. Strategies aiming to restore immunoregulation are of central importance and may be either applied directly or by changing the balance of microbiota as bacterial metabolites play a major role in intestinal immunoregulation
