Endothelial damage and dysfunction in acute graft-versus-host disease.

Clinical studies suggested that endothelial dysfunction and damage could be involved in the development and severity of acute graft-versus-host disease (aGVHD). Accordingly, we found increased percentage of apoptotic Casp3+ blood vessels in duodenal and colonic mucosa biopsies of patients with severe aGVHD. In murine experimental aGVHD, we detected severe microstructural endothelial damage and reduced endothelial pericyte coverage accompanied by reduced expression of endothelial tight junction proteins leading to increased endothelial leakage in aGVHD target organs. During intestinal aGVHD, colonic vasculature structurally changed, reflected by increased vessel branching and vessel diameter. Because recent data demonstrated an association of endothelium-related factors and steroid refractory aGVHD (SR-aGVHD), we analyzed human biopsies and murine tissues from SR-aGVHD. We found extensive tissue damage but low levels of alloreactive T cell infiltration in target organs, providing the rationale for T-cell independent SR-aGVHD treatment strategies. Consequently, we tested the endothelium-protective PDE5 inhibitor sildenafil, which reduced apoptosis and improved metabolic activity of endothelial cells in vitro. Accordingly, sildenafil treatment improved survival and reduced target organ damage during experimental SR-aGVHD. Our results demonstrate extensive damage, structural changes, and dysfunction of the vasculature during aGVHD. Therapeutic intervention by endothelium-protecting agents is an attractive approach for SR-aGVHD complementing current anti-inflammatory treatment options.

Lymphangiogenesis is a feature of acute GVHD, and VEGFR-3 inhibition protects against experimental GVHD.

Lymph vessels play a crucial role in immune reactions in health and disease. In oncology the inhibition of lymphangiogenesis is an established therapeutic concept for reducing metastatic spreading of tumor cells. During allogeneic tissue transplantation, the inhibition of lymphangiogenesis has been successfully used to attenuate graft rejection. Despite its critical importance for tumor growth, alloimmune responses, and inflammation, the role of lymphangiogenesis has not been investigated during allogeneic hematopoietic stem cell transplantation (allo-HSCT). We found that acute graft-versus-host disease (aGVHD) is associated with lymphangiogenesis in murine allo-HSCT models as well as in patient intestinal biopsies. Inhibition of aGVHD-associated lymphangiogenesis by monoclonal antibodies against vascular endothelial growth factor receptor 3 (VEGFR-3) ameliorated aGVHD and improved survival in murine models. The administration of anti-VEGFR-3 antibodies did not interfere with hematopoietic engraftment and improved immune reconstitution in allo-HSCT recipients with aGVHD. Anti-VEGFR-3 therapy had no significant impact on growth of malignant lymphoma after allo-HSCT. We conclude that aGVHD is associated with lymphangiogenesis in intestinal lesions and in lymph nodes. Our data show that anti-VEGFR-3 treatment ameliorates lethal aGVHD and identifies the lymphatic vasculature as a novel therapeutic target in the setting of allo-HSCT.

Initiation of acute graft-versus-host disease by angiogenesis.

The inhibition of inflammation-associated angiogenesis ameliorates inflammatory diseases by reducing the recruitment of tissue-infiltrating leukocytes. However, it is not known if angiogenesis has an active role during the initiation of inflammation or if it is merely a secondary effect occurring in response to stimuli by tissue-infiltrating leukocytes. Here, we show that angiogenesis precedes leukocyte infiltration in experimental models of inflammatory bowel disease and acute graft-versus-host disease (GVHD). We found that angiogenesis occurred as early as day+2 after allogeneic transplantation mainly in GVHD typical target organs skin, liver, and intestines, whereas no angiogenic changes appeared due to conditioning or syngeneic transplantation. The initiation phase of angiogenesis was not associated with classical endothelial cell (EC) activation signs, such as Vegfa/VEGFR1+2 upregulation or increased adhesion molecule expression. During early GVHD at day+2, we found significant metabolic and cytoskeleton changes in target organ ECs in gene array and proteomic analyses. These modifications have significant functional consequences as indicated by profoundly higher deformation in real-time deformability cytometry. Our results demonstrate that metabolic changes trigger alterations in cell mechanics, leading to enhanced migratory and proliferative potential of ECs during the initiation of inflammation. Our study adds evidence to the hypothesis that angiogenesis is involved in the initiation of tissue inflammation during GVHD.

Induction of HDAC2 expression upon loss of APC in colorectal tumorigenesis.

Inappropriate transcriptional repression involving histone deacetylases (HDACs) is a prominent cause for the development of leukemia. We now identify faulty expression of a specific mediator of transcriptional repression in a solid tumor. Loss of the adenomatosis polyposis coli (APC) tumor suppressor induces HDAC2 expression depending on the Wnt pathway and c-Myc. Increased HDAC2 expression is found in the majority of human colon cancer explants, as well as in intestinal mucosa and polyps of APC-deficient mice. HDAC2 is required for, and sufficient on its own to prevent, apoptosis of colonic cancer cells. Interference with HDAC2 by valproic acid largely diminishes adenoma formation in APC(min) mice. These findings point toward HDAC2 as a particularly relevant potential target in cancer therapy.

Novel pre-clinical mouse models for chronic Graft-versus-Host Disease.

Despite considerable progress in allogeneic hematopoietic cell transplantation (allo-HCT) has been achieved over the past years, chronic Graft-versus-Host Disease (cGvHD) still contributes to high morbidity rates, thus remaining a major hurdle in allo-HCT patients. To understand the complex pathophysiology of cGvHD and to develop refined prophylaxis and treatment strategies, improved pre-clinical models are needed. In this study, we developed two murine cGvHD models, which display high long-term morbidity but low mortality and depict the heterogeneous clinical manifestations of cGvHD seen in patients. We established a haploidentical C57BL/6→B6D2F1 allo-HCT model that uses myeloablative radiation and G-CSF-mobilized splenocytes as stem cell source and a sub-lethally irradiated Xenograft model, which utilizes the transfer of human peripheral blood mononuclear cells (PBMCs) into NOD scid gamma (NSG)-recipients. We characterized both mouse models to exhibit diverse clinical and histopathological signs of human cGvHD as extensive tissue damage, fibrosis/sclerosis, inflammation and B cell infiltration in cGvHD target organs skin, liver, lung and colon and found a decelerated immune cell reconstitution in the late phase after HCT. Our pre-clinical models can help to gain a deeper understanding of the target structures and mechanisms of cGvHD pathology and may enable a more reliable translation of experimental findings into the human setting of allo-HCT.

Immune Regulatory 1 Cells: A Novel and Potent Subset of Human T Regulatory Cells.

A subset of T regulatory cells (Tregs), identified by TIRC7 (T cell immune response cDNA 7) expression is designated as Immune Regulatory 1 Cells (IR1 cells). TIRC7 is an immune checkpoint inhibitor, co-localized with the T- cell receptor, HLA-DR and CTLA-4 during T-cell activation, which delivers regulatory signals via binding to its ligand, HLA-DR α2 domain. IR1 cells express FOXP3, and multiple other markers associated with immune suppression. They constitute as much as 10% of Tregs. IR1 cells strongly inhibit proliferation in mixed lymphocyte reactions, where they express high levels of IL-10. Ex vivo expansion of Tregs over 2 weeks in the presence of an agonist TIRC7 antibody disproportionately expands the IR1 Treg subset, while maintaining high expression of suppressive markers including CD39, IL-10, LAP and GARP. Ex vivo expanded IR1 cells are a potent, homogeneous, stable set of suppressor Tregs with the potential to modulate immune dysregulation. The characteristics of IR1 cells suggest a therapeutic advantage over polyclonal Tregs for therapeutic interventions. Early restoration of immune homeostasis using IR1 cells has the potential to fundamentally alter the natural history of conditions characterized by abnormalities in the T regulatory cell compartment.

Reduced Calcium Signaling Is Associated With Severe Graft-Versus-Host Disease: Results From Preclinical Models and From a Prospective EBMT Study.

Despite its involvement in various immune functions, including the allogeneic activation of T-lymphocytes, the relevance of calcium (Ca2+) for GVHD pathobiology is largely unknown. To elucidate a potential association between Ca2+and GVHD, we analyzed Ca2+-sensing G-protein coupled receptor 6a (GPRC6a) signaling in preclinical GVHD models and conducted a prospective EBMT study on Ca2+ serum levels prior alloSCT including 363 matched sibling allogeneic peripheral blood stem cell transplantations (alloSCTs). In experimental models, we found decreased Gprc6a expression during intestinal GVHD. GPRC6a deficient alloSCT recipients had higher clinical and histopathological GVHD scores leading to increased mortality. As possible underlying mechanism, we found increased antigen presentation potential in GPRC6a-/- alloSCT recipients demonstrated by higher proliferation rates of T-lymphocytes. In patients with low Ca2+ serum levels (≤median 2.2 mmol/l) before alloSCT, we found a higher incidence of acute GVHD grades II-IV (HR = 2.3 Cl = 1.45-3.85 p = 0.0006), severe acute GVHD grades III-IV (HR = 3.3 CI = 1.59-7.14, p = 0.002) and extensive chronic GVHD (HR = 2.0 Cl = 1.04-3.85 p = 0.04). In conclusion, experimental and clinical data suggest an association of reduced Ca2+ signaling with increased severity of GVHD. Future areas of interest include the in depth analysis of involved molecular pathways and the investigation of Ca2+ signaling as a therapeutic target during GVHD.

Cathepsin E Deficiency Ameliorates Graft-versus-Host Disease and Modifies Dendritic Cell Motility.

Microbial products influence immunity after allogeneic hematopoietic stem cell transplantation (allo-SCT). In this context, the role of cathepsin E (Ctse), an aspartate protease known to cleave bacterial peptides for antigen presentation in dendritic cells (DCs), has not been studied. During experimental acute graft-versus-host disease (GVHD), we found infiltration by Ctse-positive immune cells leading to higher Ctse RNA- and protein levels in target organs. In Ctse-deficient allo-SCT recipients, we found ameliorated GVHD, improved survival, and lower numbers of tissue-infiltrating DCs. Donor T cell proliferation was not different in Ctse-deficient vs. wild-type allo-SCT recipients in MHC-matched and MHC-mismatched models. Furthermore, Ctse-deficient DCs had an intact ability to induce allogeneic T cell proliferation, suggesting that its role in antigen presentation may not be the main mechanism how Ctse impacts GVHD. We found that Ctse deficiency significantly decreases DC motility in vivo, reduces adhesion to extracellular matrix (ECM), and diminishes invasion through ECM. We conclude that Ctse has a previously unrecognized role in regulating DC motility that possibly contributes to reduced DC counts and ameliorated inflammation in GVHD target organs of Ctse-deficient allo-SCT recipients. However, our data do not provide definite proof that the observed effect of Ctse-/- deficiency is exclusively mediated by DCs. A contribution of Ctse-/--mediated functions in other recipient cell types, e.g., macrophages, cannot be excluded.

Differential immunization identifies PHB1/PHB2 as blood-borne tumor antigens.

Early diagnosis of cancer is crucial for successful treatment. Noninvasive assays for detecting tumor-derived antigens in serum and other bodily fluids have the potential to screen healthy individuals for hitherto undetected cancers. Very few such assays have been successfully developed, in part because identifying potential target antigens remains a challenge. To identify new blood-borne tumor antigens for the purpose of establishing such assays, we have developed a novel technique called differential immunization. Using this method, we have identified PHB1 and PHB2, proteins thought to function as mitochondrial chaperones and transcriptional regulators, as antigens released from colorectal tumors in vivo. Serum from colorectal patients contains significantly higher levels of these antigens compared to serum from healthy volunteers. These data demonstrate that differential immunization is an effective new method for identifying tumor-derived antigens in serum.

Genes upregulated in a metastasizing human colon carcinoma cell line.

Differential gene expression between the metastatic human colon cancer cell line HT29p and its nonmetastatic counterpart HT29-MTX was revealed by suppression subtractive hybridization. Fifty-eight individual genes showed increased mRNA levels in HT29p cells. Only 15 of these genes had been related to transformation in previous studies; the majority of genes are new candidates encoding proteins relevant for the metastatic process. Cancer profiling arrays as well as in situ hybridization study revealed that at least some of the genes obtained in the SSH screen are also differentially expressed in human tumors.

Galectin-3 is strongly up-regulated in nonapoptosing mammary epithelial cells during rat mammary gland involution.

Galectin-3 is an endogenous mammalian lectin that binds to ABH carbohydrate antigens. Here we show that galectin-3 is strongly up-regulated during mammary gland involution and that it is expressed virtually exclusively on nonapoptotic cells. We demonstrate that dexamethasone, an inhibitor of the second phase of mammary gland involution, potently suppresses up-regulation of galectin-3 as judged immunohistochemically and on western blots, suggesting that systemic hormone levels regulate galectin-3 expression during involution. However, at the RNA level galectin-3 expression is rapidly up-regulated on the onset of involution but remains consistantly high during the first and second phase of involution regardless of dexamethasone treatment. These data suggest that the up-regulation of galectin-3 in the involuting mammary gland is not only controlled transcriptionally but also regulated posttranscriptionally under the control of systemic glucocorticoid hormones involved in coordinating the involution process.