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Understanding the dynamics of the immune system to cure graft-versus-host disease (GvHD).

Periodic Reporting for period 3 - GvHDCure (Understanding the dynamics of the immune system to cure graft-versus-host disease (GvHD).)

Reporting period: 2019-03-01 to 2020-08-31

"What is the problem/issue being addressed?
The major problem, that is addressed in this project, is that patients with leukemia that are treated with an allogeneic hematopoietic stem cell transplantation mainly die because of a complication called graft-versus-host disease and the recurrence of the leukemia. By using mouse models and human tissue samples we developed novel therapy strategies to improve the treatment of the complication called graft-versus-host disease and the recurrence of the leukemia.
Why is it important for society?
It is important for society to find better treatment for leukemia in order to have a healthier society and to keep more young people alive that contribute with their workforce to the wealth of the society. By distributing our results in scientific journals, by reporting them in workshops, by presenting them at scientific conferences and by publishing press releases we have informed the scientific community and the public community about our results.
What are the overall objectives?
One overall objective was to understand the role of a cell population called ""neutrophil granulocytes"" in the development of graft-versus-host disease. We were able to show that this cell type contributes to graft-versus-host disease and we found a novel pharmacological approach to target this cell type with a drug called JAK1/2 inhibitor. This objective was important because it allowed us to extend the classical type of immunosuppressive medication given to patients with graft-versus-host disease.
Another overall objective was to characterize the role of micro RNAs in the development of graft-versus-host disease. Micro RNAs are short non-coding RNAs that interfere with gene expression at the posttranscriptional level and thereby regulate multiple pathways in different diseases. We were able to show that the micro RNA miR-146a reduces the disease called graft-versus-host disease and that patients with a mutation in this micro-RNA have a higher risk to develop the disease. These results were published and are available to physicians so that they can take them into account when treating their leukemia patients.
A third overall objective was to identify new therapies for leukemia relapse. We found that inhibition of Fms-related tyrosine kinase 3 (FLT3) with sorafenib in combination with donor T cell infusions caused complete elimination of the leukemia cells in leukemia bearing mice and in patients. This observation was made in a large cohort of over 400 patients and will be an important information for physicians who treat their leukemia patients that have a FLT3-ITD mutation. This new information was also included in the guidelines for leukemia relapse treatment by the DGHO (German Society for Hematology and Oncology) which can be found at www.onkopedia.de.

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My research group, which is supported by the ERC grant, worked on the role of neutrophils in a disease called graft-versus-host disease (GVHD). The background of this project is that therapeutic interference with the early events of GVHD is difficult, and currently used immunosuppressive drugs mainly target donor T cells. However, not donor T cells but neutrophils reach the sites of tissue injury first, and therefore could be a potential target for GVHD prevention. A detailed analysis of neutrophil fate during acute GVHD and the effect on T cells is difficult because of the short lifespan of this cell type. We could show in this reporting period that neutrophils that had been photoconverted in the a section of the intestinal tract called ileum later migrated to lymph nodes located in close proximity to the intestines. We also observed that the neutrophils colocalized with T cells and presented antigen on major histocompatibility complex (MHC)-II, thereby affecting T cell expansion. We found that JAK1/JAK2 inhibition with a drug called ruxolitinib reduced neutrophil influx into the lymph nodes and acute GVHD pathogenesis. In agreement with this finding, neutrophil depletion reduced acute GVHD. We conclude that neutrophils are attracted to the ileum, where the intestinal barrier is disrupted, and then migrate to the mLN, where they participate in alloantigen presentation.
Another major work package was the analysis of the role of micro-RNAs in GVHD. We could show that miR-146a plays a crucial role in GVHD. We found that patients with a G/C polymorphism within the human microRNA-146a (miR-146a) gene was strongly associated with the risk of developing severe acute GVHD. In mice, deficiency of miR-146a in the hematopoietic system or transfer of recipient-type miR-146a-/- dendritic cells (DCs) enhanced GVHD, while miR-146a mimic-transfected DCs ameliorated disease. Mechanistically, lack of miR-146a enhanced JAK2-STAT1 pathway activity, which led to higher expression of class II-transactivator (CIITA) and consecutively increased MHCII-levels on DCs. Inhibition of JAK1/2 or CIITA knockdown in DCs prevented miR-146a-/- DC-induced GVHD exacerbation. Consistent with our findings in mice, patients with the miR-146a polymorphism rs2910164 in hematopoietic cells displayed higher MHCII levels on monocytes, which could be targeted by JAK1/2 inhibition. Our findings indicate that the miR-146a polymorphism rs2910164 identifies patients at high risk for GVHD before allo-HCT. Functionally we show that miR-146a acts as a central regulator of recipient-type DC activation during GVHD by dampening the pro-inflammatory JAK-STAT/CIITA/MHCII axis, which provides a scientific rationale for early JAK1/2 inhibition in selected patients.
To understand the role of Caveolin-1 (Cav-1) as a key organizer of membrane specializations and a scaffold protein that regulates signaling in multiple cell types in acute GVHD, we used Cav-1 deficient mice as donors for the transferred T cells. We found that increased Cav-1 expression in human and murine T cells after allogeneic hematopoietic cell transplantation. Indeed, Cav-1(-/-) donor T cells caused less severe acute graft-versus-host disease (GVHD) and yielded higher numbers of regulatory T cells (Tregs) compared with controls. Depletion of Tregs from the graft abrogated this protective effect. Correspondingly, Treg frequencies increased when Cav-1(-/-) T cells were exposed to transforming growth factor-β/T-cell receptor (TCR)/CD28 activation or alloantigen stimulation in vitro compared with wild-type T cells. Mechanistically, we found that the phosphorylation of Cav-1 is dispensable for the control of T-cell fate by using a nonphosphorylatable Cav-1 (Y14F/Y14F) point-mutation variant. Moreover, the close proximity of lymphocyte-specific protein tyrosine kinase (Lck) to the TCR induced by TCR-activation was reduced in Cav-1(-/-)T cells. Therefore, less TCR/Lck clustering results in suboptimal activation of the downstream signaling events, which correlates with the preferential development into a Treg phenotype. Overall, we report a novel role for Cav-1 in TCR/Lck spatial distribution upon TCR triggering, which controls T-cell fate toward a regulatory phenotype. This alteration translated into a significant increase in the frequency of Tregs and reduced GVHD in vivo.
Pathogenic kinase sinaling is not only found in immune cells that cause GVHD but also in leukemia cells that cause relapse after allo-HCT. Individuals with acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) in the gene encoding Fms-related tyrosine kinase 3 (FLT3) who relapse after allogeneic hematopoietic cell transplantation (allo-HCT) have a 1-year survival rate below 20%. We observed that sorafenib, a multitargeted tyrosine kinase inhibitor, increased IL-15 production by FLT3-ITD+ leukemia cells. This synergized with the allogeneic CD8+ T cell response, leading to long-term survival in six mouse models of FLT3-ITD+ AML. Sorafenib-related IL-15 production caused an increase in CD8+CD107a+IFN-γ+ T cells with features of longevity (high levels of Bcl-2 and reduced PD-1 levels), which eradicated leukemia in secondary recipients. Mechanistically, sorafenib reduced expression of the transcription factor ATF4, thereby blocking negative regulation of interferon regulatory factor 7 (IRF7) activation, which enhanced IL-15 transcription. Both IRF7 knockdown and ATF4 overexpression in leukemia cells antagonized sorafenib-induced IL-15 production in vitro. Human FLT3-ITD+ AML cells obtained from sorafenib responders following sorafenib therapy showed increased levels of IL-15, phosphorylated IRF7, and a transcriptionally active IRF7 chromatin state. The mitochondrial spare respiratory capacity and glycolytic capacity of CD8+ T cells increased upon sorafenib treatment in sorafenib responders but not in nonresponders. Our findings indicate that the synergism of T cells and sorafenib is mediated via reduced ATF4 expression, causing activation of the IRF7-IL-15 axis in leukemia cells and thereby leading to metabolic reprogramming of leukemia-reactive T cells in humans. Therefore, sorafenib treatment has the potential to contribute to an immune-mediated cure of FLT3-ITD-mutant AML relapse, an otherwise fatal complication after allo-HCT.
A major progress beyond the state of the art was that we could show a novel role for neutrophil granulocytes in antigen presentation in acute GVHD. We will build on this finding and characterize more pro- and anti-inflammatory events that influence neutrophil migration and function during GVHD. We expect that we can characterize different subtypes of neutrophils that are involved in the early versus late phase of GVHD. We were able to generate preliminary by single cell sorting and RNA sequencing of neutrophils from the intestinal tract of mice developing GVHD. Our findings go beyond the state of the art as there is currently no information which neutrophil subtypes reside in the intestinal tract at different time points after transplantation. Another major discovery was that neutrophils mediate cellular communication between the inflamed tissue and the secondary lymphoid organs during GVHD development. This was a major novel concept as classically neutrophils had been considered as short lived cells that cannot perform reverse migration from the inflamed target organ to lymph nodes where T cell priming takes place. In future studies we plan to characterize the signaling events that take place in neutrophils.
In order to extend our finding that miR-146a plays a central role in GVHD we will screen for other micro RNAs that could play a role in GVHD. To improve strategies that enhance the graft-versus-leukemia effect we will combine T cell transfer with kinase inhibitors that may promote the immune response against leukemia cells after allogeneic hematopoietic stem cell transplantation.
We expect to archive a better understanding of several aspects of GVHD pathogenesis until the end of the funding period. In particular we will study the role of different types of innate immune cells during early events of GVHD, the role of ER stress in GVHD and tissue damage and the impact of micro RNAs in different cellular compartments during GVHD. To attain a deeper understanding of the biology of graft-versus-leukemia effects we plan to analyze different signaling pathways in leukemia cells and exploit the insight to develop novel therapeutic strategies that block leukemia cell survival while enhancing the T cell immune response against leukemia antigens.