Analysis of the Immune Cell Infiltrates and Biomarkers during acute Gastrointestinal Graft vs Host Disease

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

Pathopysiology of GvHD and HSCT related complications

For over 60 years, hematopoietic stem cell transplantation (HSCT) has been the major curative therapy for several hematological and genetic disorders, but its efficacy is limited by the secondary disease called graft versus host disease (GvHD). Huge advances have been made in successful transplantation in order to improve patient quality of life and yet, complete success is hard to achieve. This review assimilates recent updates on pathophysiology of GvHD, prophylaxis and treatment of GvHD related complications and advances in the potential treatment of GvHD

GPR Expression in Intestinal Biopsies From SCT Patients Is Upregulated in GvHD and Is Suppressed by Broad-Spectrum Antibiotics

Microbiota can exert immunomodulatory effects by short-chain fatty acids (SCFA) in experimental models of graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-SCT). Therefore we aimed to analyze the expression of SCFAs sensing G-protein coupled receptor GPR109A and GPR43 by quantitative PCR in 338 gastrointestinal (GI) biopsies obtained from 199 adult patients undergoing allo-SCT and assessed the interaction of GPR with FOXP3 expression and regulatory T cell infiltrates. GPR expression was strongly upregulated in patients with stage II-IV GvHD (p=0.000 for GPR109A, p=0.01 for GPR43) and at the onset of GvHD (p 0.000 for GPR109A, p=0.006 for GPR43) and correlated strongly with FOXP3 and NLRP3 expression. The use of broad-spectrum antibiotics (Abx) drastically suppressed GPR expression as well as FOXP3 expression in patients’ gut biopsies (p=0.000 for GPRs, FOXP3 mRNA and FOXP3+ cellular infiltrates). Logistic regression analysis revealed treatment with Abx as an independent factor associated with GPR and FOXP3 loss. The upregulation of GPRs was evident only in the absence of Abx (p=0.001 for GPR109A, p=0.014 for GPR43) at GvHD onset. Thus, GPR expression seems to be upregulated in the presence of commensal bacteria and associates with infiltration of FOXP3+ T regs, suggesting a protective, regenerative immunomodulatory response. However, Abx, which has been shown to induce dysbiosis, interferes with this protective response

Downregulation of the vitamin D receptor expression during acute gastrointestinal graft versus host disease is associated with poor outcome after allogeneic stem cell transplantation

The vitamin D receptor (VDR) is critical in regulating intestinal homeostasis and emerging evidence demonstrates that VDR deficiency is a critical factor in inflammatory bowel disease pathology. However, no clinical data exist regarding the intestinal expression of VDR in patients after allogeneic haematopoietic stem cell transplantation (HSCT). Analyzing intestinal biopsies from 90 patients undergoing HSCT with mortality follow-up, we demonstrated that patients with severe acute gastrointestinal graft versus host disease (GIGvHD) showed significant downregulation of VDR gene expression compared to mild or no acute GI-GvHD patients (p = 0.007). Reduced VDR expression was already detectable at acute GI-GvHD onset compared to GvHD-free patients (p = 0.01). These results were confirmed by immunohistochemistry (IHC) where patients with severe acute GI-GvHD showed fewer VDR+ cells (p =0.03) and a reduced VDR staining score (p = 0.02) as compared to mild or no acute GI-GvHD patients. Accordingly, low VDR gene expression was associated with a higher cumulative incidence of treatment-related mortality (TRM) (p =1.6x10-6) but not with relapse-related mortality (RRM). A multivariate Cox regression analysis identified low VDR as an independent risk factor for TRM (p = 0.001, hazard ratio 4.14, 95% CI 1.78-9.63). Furthermore, VDR gene expression significantly correlated with anti-microbial peptides (AMPs) gene expression (DEFA5: r = 0.637, p = 7x10-5, DEFA6: r 0 0.546, p = 0.001). In conclusion, our findings suggest an essential role of the VDR in the pathogenesis of gut GvHD and the prognosis of patients undergoing HSCT

Anti-Thymocyte Globulin Treatment Augments 1,25-Dihydroxyvitamin D3 Serum Levels in Patients Undergoing Hematopoietic Stem Cell Transplantation

Application of anti-thymocyte globulin (ATG) is a widely used strategy for the prevention of graftversus-host disease (GvHD). As vitamin D3 serum levels are also discussed to affect hematopoietic stem cell transplantation (HSCT) outcome and GvHD development, we analysed a possible interplay between ATG treatment and serum levels of 25-hydroxyvitamin D3and 1,25-dihydroxyvitaminD3in 4HSCT cohorts withdifferent vitaminD3supplementation. ATG is significantly associated with higher serum level of 1,25 dihydroxyvitamin D3 around HSCT (day -2 to 7, peri-transplant), however only in patients with adequate levels of its precursor 25-hydroxyvitamin D3. ATG exposure had no impact on overall survival in patients supplemented with high dose vitamin D3, but was associated with higher risk of one-year treatment-related mortality (log rank test p=0.041) in patients with no/low vitamin D3 supplementation. However, the difference failed to reach significance applying a Cox-model regression without and with adjustment for baseline risk factors (unadjusted P=0,058, adjusted p=0,139). To shed some light on underlying mechanisms, we investigated the impact of ATG on 1,25-DihydroxyvitaminD3 production by human dendritic cells (DCs) in vitro.ATGincreased gene expression ofCYP27B1, the enzyme responsible for the conversion of 25-hydroxyvitamin D3 into 1,25-dihydroxyvitamin D3, which was accompanied by higher 1,25-dihydroxyvitamin D3levels in ATG-treatedDCculture supernatants.Our data demonstrate a cooperative effect of 25-hydroxyvitamin D3 and ATG in the regulation of 1,25-dihydroxyvitamin D3 production. This finding may be of importance in the context of HSCT, where early high levels of 1,25- dihydroxyvitamin D3 levels have been shown to be predictive for lower transplant related mortality and suggest that vitamin D3 supplementation may especially be important in patients receiving ATG for GvHD prophylaxis

Reg3α concentrations at day of allogeneic stem cell transplantation predict outcome and correlate with early antibiotic use

Intestinal microbiome diversity plays an important role in the pathophysiology of acute gastrointestinal (GI) graft-versus-host disease (GVHD) and influences the outcome of patients after allogeneic stem cell transplantation (ASCT). We analyzed clinical data and blood samples taken preconditioning and on the day of ASCT from 587 patients from 7 German centers of the Mount Sinai Acute GVHD International Consortium, dividing them into single-center test (n = 371) and multicenter validation (n = 216) cohorts. Regenerating islet–derived 3α (Reg3α) serum concentration of day 0 correlated with clinical data as well as urinary 3-indoxylsulfate (3-IS) and Clostridiales group XIVa, indicators of intestinal microbiome diversity. High Reg3α concentration at day 0 of ASCT was associated with higher 1-year transplant-related mortality (TRM) in both cohorts (P < .001). Cox regression analysis revealed high Reg3α at day 0 as an independent prognostic factor for 1-year TRM. Multivariable analysis showed an independent correlation of high Reg3α concentrations at day 0 with early systemic antibiotic (AB) treatment. Urinary 3-IS (P = .04) and Clostridiales group XIVa (P = .004) were lower in patients with high vs those with low day 0 Reg3α concentrations. In contrast, Reg3α concentrations before conditioning therapy correlated neither with TRM nor disease or treatment-related parameters. Reg3α, a known biomarker of acute GI GVHD correlates with intestinal dysbiosis, induced by early AB treatment in the period of pretransplant conditioning. Serum concentrations of Reg3α measured on the day of graft infusion are predictive of the risk for TRM of ASCT recipients

Indoxyl 3-sulfate inhibits maturation and activation of human monocyte-derived dendritic cells

Indole is produced from L-tryptophan by commensal bacteria and further metabolized to indoxyl 3-sulfate (I3S) in the liver. Physiologic concentrations of I3S are related to a lower risk to develop graft versus host disease in allogeneic stem cell transplanted patients pointing towards an immunoregulatory function of I3S. Here we investigated the impact of I3S on the maturation of human monocyte-derived dendritic cells (DCs). Even pathophysiologic concentrations of I3S did not affect viability of mature DCs, but I3S decreased the expression of co-stimulatory molecules such as CD80 and CD86 on mature DCs. Furthermore, I3S inhibited IL-12 and IL-6 secretion by mature DCs while IL-10 was significantly upregulated. Co-culture of I3S-treated mature DCs with allogeneic T cells revealed no alteration in T cell proliferation. However, interferon gamma and TNF production of T cells was suppressed. As I3S exerted no direct effect on T cells, the defect in T cell activation was mediated by I3S-treated mature DCs. Our study suggests an anti-inflammatory and tolerizing effect of I3S on human DCs

Immune Reconstitution after Allogeneic Hematopoietic Stem Cell Transplantation

The reconstitution and the regaining of function of a healthy, transplanted immune system is of utmost importance for the recovery and long term survival of patients after HSCT. New developments within HSCT, for example, umbilical cord blood or haploidentical grafts, both leading to prolonged immunodeficiency and delayed immune reconstitution, have increased the need to improve immune reconstitution. Thus, understanding and enhancing immune reconstitution post-HSCT is an area of intense research. Immune reconstitution post-HSCT occurs in several steps, innate immunity being the first to regain function. Although the slow T-cell reconstitution is regarded as primarily responsible for deleterious infections with latent viruses or fungi, occurrence of graft-versus-host disease, and relapse, the importance of innate immune cells for disease and infection control is being reevaluated. Here we aim to summarize the major steps of the immune reconstitution in patients after HSCT

Non-invasive diagnosis of acute intestinal graft-versus-host disease by a new scoring system using ultrasound morphology, compound elastography, and contrast-enhanced ultrasound

Acute gastrointestinal (GI) graft-versus-host disease (GvHD) is a life-threating complication in patients after allogeneic stem cell transplantation (ASCT). In 60 sonographic analyses, a novel scoring system for non-invasive diagnosis of severe GI GvHD was developed. The score comprised morphological and vascular changes using B-mode and color-coded Doppler sonography, changes of mural stiffness using compound elastography, and dynamic microvascularisation using contrast-enhanced ultrasound (CEUS). Furthermore, inflammatory parameters such as CRP, Calprotectin, and regenerating islet-derived protein 3 alpha (Reg3 alpha) were obtained. ROC curve analysis of our novel GvHD sum score revealed an area under the curve of 1.0 (95% CI: 0.99-1.00) in diagnosing GI GvHD and 0.88 (95% CI: 0.79-0.96) for severe GI GvHD. A sum score above 5 correlated with GI GvHD with a sensitivity of 97.6% (41/42) and a specificity of 94.4% (17/18) and score values above 10 with severe GI GvHD with a sensitivity of 91.7% (11/12) and specificity of 79.2% (38/48). The additional use of inflammatory parameters did not improve the predictive power. CEUS is a promising, non-invasive tool for the diagnosis of acute GI GvHD. Together with further descriptive parameters for inflammatory processes, it gains significant diagnostic accuracy in identifying patients with severe stages of acute intestinal GvHD