Der Einfluss von plasmazytoiden dendritischen Zellen und immunstimulatorischer DNA auf B-Zellen des humanen Immunsystems

It has been reported that interferon (IFN-) enhances humoral immunity and that dendritic cells of the myeloid lineage promote B-cell differentiation. Here we studied whether the plasmacytoid dendritic cell (PDC), a subset of dendritic cells specialized for the production of IFN-, is involved in regulating B-cell differentiation and immunoglobulin production. The recently identified class of CpG oligonucleotides (CpG-C) was used to activate both B cells and PDCs via Toll-like receptor 9 (TLR9). The presence of PDCs synergistically enhanced CD86 expression, cytokine production (interleukin 6 [IL-6], tumor necrosis factor , and IL-10) and plasma cell differentiation of isolated human peripheral blood B cells stimulated through CpG-C and B-cell antigen receptor (BCR) ligation. This stimulation protocol was sufficient to drive purified naive B cells into IgM-producing plasma cells and to trigger IgG synthesis in memory B cells. PDCs contributed to B-cell activation via IFN- secretion. Up-regulation of TLR9 on B cells was not involved. These results demonstrate that CpG-stimulated PDCs induce plasma cell differentiation in naive and memory B cells in the absence of T-cell help, providing an explanation for the excellent activity of CpG oligonucleotides as a humoral vaccine adjuvant. (Blood. 2004;103:3058-3064

Novel T cell/organoid culture system allows ex vivo modeling of intestinal graft-versus-host disease

Acute graft-versus-host disease (GvHD) remains the biggest clinical challenge and prognosis-determining complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Donor T cells are acceptedly key mediators of alloreactivity against host tissues and here especially the gut. In support of previous studies, we found that the intestinal intra-epithelial lymphocyte (IEL) compartment was dynamically regulated in the course of MHC class I full mismatch allo-HSCT. However, while intestinal epithelial cell (IEC) damage endangers the integrity of the intestinal barrier and is a core signature of intestinal GvHD, the question whether and to what degree IELs are contributing to IEC dysregulation is poorly understood. To study lymphoepithelial interaction, we employed a novel ex vivo T cell/organoid co-culture model system. Here, allogeneic intra-epithelial T cells were superior in inducing IEC death compared to syngeneic IEL and allogeneic non-IEL T cells. The ability to induce IEC death was predominately confined to TCRβ+ T cells and was executed in a largely IFNγ-dependent manner. Alloreactivity required a diverse T cell receptor (TCR) repertoire since IELs genetically modified to express a TCR restricted to a single, non-endogenous antigen failed to mediate IEC pathology. Interestingly, minor histocompatibility antigen (miHA) mismatch was sufficient to elicit IEL-driven IEC damage. Finally, advanced live cell imaging analyses uncovered that alloreactive IELs patrolled smaller areas within intestinal organoids compared to syngeneic controls, indicating their unique migratory properties within allogeneic IECs. Together, we provide here experimental evidence for the utility of a co-culture system to model the cellular and molecular characteristics of the crosstalk between IELs and IEC in an allogeneic setting ex vivo. In the light of the emerging concept of dysregulated immune-epithelial homeostasis as a core aspect of intestinal GvHD, this approach represents a novel experimental system to e.g. screen therapeutic strategies for their potential to normalize T cell/IEC- interaction. Hence, analyses in pre-clinical in vivo allo-HSCT model systems may be restricted to hereby positively selected, promising approaches

Selection of molecular structure and delivery of RNA oligonucleotides to activate TLR7 versus TLR8 and to induce high amounts of IL-12p70 in primary human monocytes

Detection of non-self RNA by TLRs within endosomes and by retinoic acid-inducible gene I (RIG-I)-like helicases in the cytosol is central to mammalian antiviral immunity. In this study, we used pathway-specific agonists and targeted delivery to address RNA immunorecognition in primary human immune cells. Within PBMC, plasmacytoid dendritic cells (pDC) and monocytes were found to be responsible for IFN-alpha production upon immunorecognition of RNA. The mechanisms of RNA recognition in pDC and monocytes were distinct. In pDC, recognition of ssRNA and dsRNA oligonucleotides was TLR7-dependent, whereas a 5' triphosphate moiety (RIG-I ligand activity) had no major contribution to IFN-alpha production. In monocytes, the response to RNA oligonucleotides was mediated by either TLR8 or RIG-I. TLR8 was responsible for IL-12 induction upon endosomal delivery of ssRNA oligonucleotides and RIG-I was responsible for IFN-alpha production upon delivery of 5' triphosphate RNA into the cytosol. In conclusion, the dissection of these pathways by selecting the appropriate structure and delivery of RNA reveals pDC as major producer of IFN-alpha upon TLR-mediated stimulation and monocytes as major producer of IFN-alpha upon RIG-I-mediated stimulation. Furthermore, our results uncover the potential of monocytes to function as major producers of IL-12p70, a key Th1 cytokine classically ascribed to myeloid dendritic cells that cannot be induced by CpG oligonucleotides in the human system

The microbial metabolite desaminotyrosine enhances T-cell priming and cancer immunotherapy with immune checkpoint inhibitors

Background Inter-individual differences in response to immune checkpoint inhibitors (ICI) remain a major challenge in cancer treatment. The composition of the gut microbiome has been associated with differential ICI outcome, but the underlying molecular mechanisms remain unclear, and therapeutic modulation challenging. Methods We established an in vivo model to treat C57Bl/6j mice with the type-I interferon (IFN-I)-modulating, bacterial-derived metabolite desaminotyrosine (DAT) to improve ICI therapy. Broad spectrum antibiotics were used to mimic gut microbial dysbiosis and associated ICI resistance. We utilized genetic mouse models to address the role of host IFN-I in DAT-modulated antitumour immunity. Changes in gut microbiota were assessed using 16S-rRNA sequencing analyses. Findings We found that oral supplementation of mice with the microbial metabolite DAT delays tumour growth and promotes ICI immunotherapy with anti-CTLA-4 or anti-PD-1. DAT-enhanced antitumour immunity was associated with more activated T cells and natural killer cells in the tumour microenvironment and was dependent on host IFN-I signalling. Consistent with this, DAT potently enhanced expansion of antigen-specific T cells following vaccination with an IFN-I-inducing adjuvant. DAT supplementation in mice compensated for the negative effects of broad-spectrum antibiotic-induced dysbiosis on anti-CTLA-4-mediated antitumour immunity. Oral administration of DAT altered the gut microbial composition in mice with increased abundance of bacterial taxa that are associated with beneficial response to ICI immunotherapy. Interpretation We introduce the therapeutic use of an IFN-I-modulating bacterial-derived metabolite to overcome resistance to ICI. This approach is a promising strategy particularly for patients with a history of broad-spectrum antibiotic use and associated loss of gut microbial diversity

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

Coronavirus disease 2019 induces multi‐lineage, morphologic changes in peripheral blood cells

The clinical course of coronavirus disease 2019 (COVID‐19) varies from mild symptoms to acute respiratory distress syndrome, hyperinflammation, and coagulation disorder. The hematopoietic system plays a critical role in the observed hyperinflammation, particularly in severely ill patients. We conducted a prospective diagnostic study performing a blood differential analyzing morphologic changes in peripheral blood of COVID‐19 patients. COVID‐19 associated morphologic changes were defined in a training cohort and subsequently validated in a second cohort (n = 45). Morphologic aberrations were further analyzed by electron microscopy (EM) and flow cytometry of lymphocytes was performed. We included 45 COVID‐19 patients in our study (median age 58 years; 82% on intensive care unit). The blood differential showed a specific pattern of pronounced multi‐lineage aberrations in lymphocytes (80%) and monocytes (91%) of patients. Overall, 84%, 98%, and 98% exhibited aberrations in granulopoiesis, erythropoiesis, and thrombopoiesis, respectively. Electron microscopy revealed the ultrastructural equivalents of the observed changes and confirmed the multi‐lineage aberrations already seen by light microscopy. The morphologic pattern caused by COVID‐19 is characteristic and underlines the serious perturbation of the hematopoietic system. We defined a hematologic COVID‐19 pattern to facilitate further independent diagnostic analysis and to investigate the impact on the hematologic system during the clinical course of COVID‐19 patients

The Nlrp3 inflammasome regulates acute graft-versus-host disease

The success of allogeneic hematopoietic cell transplantation is limited by acute graft-versus-host disease (GvHD), a severe complication accompanied by high mortality rates. Yet, the molecular mechanisms initiating this disease remain poorly defined. In this study, we show that, after conditioning therapy, intestinal commensal bacteria and the damage-associated molecular pattern uric acid contribute to Nlrp3 inflammasome-mediated IL-1β production and that gastrointestinal decontamination and uric acid depletion reduced GvHD severity. Early blockade of IL-1β or genetic deficiency of the IL-1 receptor in dendritic cells (DCs) and T cells improved survival. The Nlrp3 inflammasome components Nlrp3 and Asc, which are required for pro-IL-1β cleavage, were critical for the full manifestation of GvHD. In transplanted mice, IL-1β originated from multiple intestinal cell compartments and exerted its effects on DCs and T cells, the latter being preferentially skewed toward Th17. Compatible with these mouse data, increased levels of active caspase-1 and IL-1β were found in circulating leukocytes and intestinal GvHD lesions of patients. Thus, the identification of a crucial role for the Nlrp3 inflammasome sheds new light on the pathogenesis of GvHD and opens a potential new avenue for the targeted therapy of this severe complication

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

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

Severe T cell hyporeactivity in ventilated COVID-19 patients correlates with prolonged virus persistence and poor outcomes

Coronavirus disease 2019 (COVID-19) can lead to pneumonia and hyperinflammation. Here we show a sensitive method to measure polyclonal T cell activation by downstream effects on responder cells like basophils, plasmacytoid dendritic cells, monocytes and neutrophils in whole blood. We report a clear T cell hyporeactivity in hospitalized COVID-19 patients that is pronounced in ventilated patients, associated with prolonged virus persistence and reversible with clinical recovery. COVID-19-induced T cell hyporeactivity is T cell extrinsic and caused by plasma components, independent of occasional immunosuppressive medication of the patients. Monocytes respond stronger in males than females and IL-2 partially restores T cell activation. Downstream markers of T cell hyporeactivity are also visible in fresh blood samples of ventilated patients. Based on our data we developed a score to predict fatal outcomes and identify patients that may benefit from strategies to overcome T cell hyporeactivity.Coronavirus disease 2019 (COVID-19) can lead to pneumonia and hyperinflammation. Here we show a sensitive method to measure polyclonal T cell activation by downstream effects on responder cells like basophils, plasmacytoid dendritic cells, monocytes and neutrophils in whole blood. We report a clear T cell hyporeactivity in hospitalized COVID-19 patients that is pronounced in ventilated patients, associated with prolonged virus persistence and reversible with clinical recovery. COVID-19-induced T cell hyporeactivity is T cell extrinsic and caused by plasma components, independent of occasional immunosuppressive medication of the patients. Monocytes respond stronger in males than females and IL-2 partially restores T cell activation. Downstream markers of T cell hyporeactivity are also visible in fresh blood samples of ventilated patients. Based on our data we developed a score to predict fatal outcomes and identify patients that may benefit from strategies to overcome T cell hyporeactivity