Annette Schmitt-Graeff

Activity of therapeutic JAK 1/2 blockade in graft-versus-host disease

Graft-versus-host-disease (GVHD) is a severe complication of allogeneic hematopoietic cell transplantation (allo-HCT) characterized by the production of high levels of proinflammatory cytokines. Activated Janus kinases (JAKs) are required for T-effector cell responses in different inflammatory diseases, and their blockade could potently reduce acute GVHD. We observed that inhibition of JAK1/2 signaling resulted in reduced proliferation of effector T cells and suppression of proinflammatory cytokine production in response to alloantigen in mice. In vivo JAK 1/2 inhibition improved survival of mice developing acute GVHD and reduced histopathological GVHD grading, serum levels of proinflammatory cytokines, and expansion of alloreactive luc-transgenic T cells. Mechanistically, we could show that ruxolitinib impaired differentiation of CD4(+) T cells into IFN-γ- and IL17A-producing cells, and that both T-cell phenotypes are linked to GVHD. Conversely, ruxolitinib treatment in allo-HCT recipients increased FoxP3(+) regulatory T cells, which are linked to immunologic tolerance. Based on these results, we treated 6 patients with steroid-refractory GVHD with ruxolitinib. All patients responded with respect to clinical GVHD symptoms and serum levels of proinflammatory cytokines. In summary, ruxolitinib represents a novel targeted approach in GVHD by suppression of proinflammatory signaling that mediates tissue damage and by promotion of tolerogenic Treg cells.

Antiviral and Regulatory T Cell Immunity in a Patient with Stromal Interaction Molecule 1 Deficiency

Stromal interaction molecule 1 (STIM1) deficiency is a rare genetic disorder of store-operated calcium entry, associated with a complex syndrome including immunodeficiency and immune dysregulation. The link from the molecular defect to these clinical manifestations is incompletely understood. We report two patients with a homozygous R429C point mutation in STIM1 completely abolishing store-operated calcium entry in T cells. Immunological analysis of one patient revealed that despite the expected defect of T cell proliferation and cytokine production in vitro, significant antiviral T cell populations were generated in vivo. These T cells proliferated in response to viral Ags and showed normal antiviral cytotoxicity. However, antiviral immunity was insufficient to prevent chronic CMV and EBV infections with a possible contribution of impaired NK cell function and a lack of NKT cells. Furthermore, autoimmune cytopenia, eczema, and intermittent diarrhea suggested impaired immune regulation. FOXP3-positive regulatory T (Treg) cells were present but showed an abnormal phenotype. The suppressive function of STIM1-deficient Treg cells in vitro, however, was normal. Given these partial defects in cytotoxic and Treg cell function, impairment of other immune cell populations probably contributes more to the pathogenesis of immunodeficiency and autoimmunity in STIM1 deficiency than previously appreciated.

The IL-33/ST2 axis augments effector T cell responses during acute GVHD

Interleukin (IL)-33 binding to the receptor suppression of tumorigenicity 2 (ST2) produces pro-inflammatory and anti-inflammatory effects. Increased levels of soluble ST2 (sST2) are a biomarker for steroid-refractory graft-versus-host disease (GVHD) and mortality. However, whether sST2 has a role as an immune modulator or only as a biomarker during GVHD was unclear. We show increased IL-33 production by nonhematopoietic cells in the gastrointestinal (GI) tract in mice post-conditioning and patients during GVHD. Exogenous IL-33 administration during the peak inflammatory response worsened GVHD. Conversely, GVHD lethality and tumor necrosis factor-α production was significantly reduced in il33(-/-) recipients. ST2 was upregulated on murine and human alloreactive T cells and sST2 increased as experimental GVHD progressed. Concordantly, st2(-/-) vs wild-type (WT) donor T cells had a marked reduction in GVHD lethality and GI histopathology. Alloantigen-induced IL-18 receptor upregulation was lower in st2(-/-) T cells, and linked to reduced interferon-γ production by st2(-/-) vs WT T cells during GVHD. Blockade of IL-33/ST2 interactions during allogeneic-hematopoietic cell transplantation by exogenous ST2-Fc infusions had a marked reduction in GVHD lethality, indicating a role of ST2 as a decoy receptor modulating GVHD. Together, these studies point to the IL-33/ST2 axis as a novel and potent target for GVHD therapy.

Human metapneumovirus induces more severe disease and stronger innate immune response in BALB/c mice as compared with respiratory syncytial virus

BackgroundHuman metapneumovirus (HMPV) and respiratory syncytial virus (RSV) are members of the Pneumovirinae subfamily of Paramyxoviridae and can cause severe respiratory disease, especially in infants and young children. Some differences in the clinical course of these infections have been described, but there are few comparative data on pathogenesis in humans and animal models. In this study, HMPV and RSV were compared for replication, pathogenesis and immune induction in BALB/c mice infected with equivalent inocula of either virus.MethodsViral titers in the lungs and in the nasal turbinates of mice were determined by plaque assay. Histopathological changes in the lungs as well as weight loss and levels of airway obstruction were monitored in the infected mice to record the severity of illness. Inflammatory cells recruited to the lungs were characterized by flow cytometry and by differential staining. In the case of natural killer cells, cytotoxic activity was also measured. Cytokine levels in the BAL were determined by cytometric bead array.ResultsRSV replicated to higher titers than HMPV in the lung and in the upper respiratory tract (URT), and virus elimination from the lungs was more rapid in HMPV-infected mice. Clinical illness as determined by airway obstruction, weight loss, and histopathology was significantly more severe after HMPV infection. A comparison of the cellular immune response revealed similar recruitment of T lymphocytes with a predominance of IFN-γ-producing CD8+ T cells. By contrast, there were obvious differences in the innate immune response. After HMPV infection, more neutrophils could be detected in the airways and there were more activated NK cells than in RSV-infected mice. This correlated with higher levels of IL-6, TNF-α and MCP-1.ConclusionThis study shows important differences in HMPV and RSV pathogenesis and suggests that the pronounced innate immune response observed after HMPV infection might be instrumental in the severe pathology.

A Novel Function for P2Y2 in Myeloid Recipient–Derived Cells during Graft-versus-Host Disease

Acute graft-versus-host disease (GvHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation. During the initiation phase of acute GvHD, endogenous danger signals such as ATP are released and inform the innate immune system via activation of the purinergic receptor P2X7 that a noninfectious damage has occurred. A second ATP-activated purinergic receptor involved in inflammatory diseases is P2Y2. In this study, we used P2y2−/− mice to test the role of this receptor in GvHD. P2y2−/− recipients experienced reduced GvHD-related mortality, IL-6 levels, enterocyte apoptosis, and histopathology scores. Chimeric mice with P2y2 deficiency restricted to hematopoietic tissues survived longer after GvHD induction than did wild-type mice. P2y2 deficiency of the recipient was connected to lower levels of myeloperoxidase in the intestinal tract of mice developing GvHD and a reduced myeloid cell signature. Selective deficiency of P2Y2 in inflammatory monocytes decreased GvHD severity. Mechanistically, P2y2−/− inflammatory monocytes displayed defective ERK activation and reactive oxygen species production. Compatible with a role of P2Y2 in human GvHD, the frequency of P2Y2+ cells in inflamed GvHD lesions correlated with histopathological GvHD severity. Our findings indicate a novel function for P2Y2 in ATP-activated recipient myeloid cells during GvHD, which could be exploited when targeting danger signals to prevent GvHD.

Consensus on the histopathological evaluation of liver biopsies from patients following allogeneic hematopoietic cell transplantation

After allogeneic hematopoietic cell transplantation (alloHCT) liver biopsy is performed for enigmatic liver disorders when noninvasive diagnostic steps have failed in establishing a definitive diagnosis. This document provides an updated consensus on the prerequisites for proper evaluation of liver biopsies in alloHCT patients and the histological diagnostic criteria for liver graft-versus-host disease (GvHD). The Working Group’s recommendations for the histological diagnosis of liver GvHD were derived from the peer-reviewed literature and from the consensus diagnosis of a total of 30 coded liver biopsies. Acceptance of the recommendations was tested by a survey distributed to all HCT centers in Austria, Germany and Switzerland. Consensus was achieved for biopsy indications, methods of sample acquisition and processing, reporting and interpretation of biopsy findings. As GvHD is variably treated and the treatment modalities have changed over time, the panel endorses the use of more frequent biopsies in clinical studies in order to improve the present challenging clinical and diagnostic situation.

Ill-Defined Germinal Centers and Severely Reduced Plasma Cells are Histological Hallmarks of Lymphadenopathy in Patients with Common Variable Immunodeficiency

Given the severely reduced numbers of circulating class-switched memory B cells and plasmablasts in patients with common variable immunodeficiency (CVID) the germinal center (GC) reaction as the source of both populations is expected to be disturbed in many CVID patients. Therefore immunohistochemical studies were performed on lymph node (LN) biopsies from ten CVID patients with benign lymphoproliferation. According to the Sander classification the majority of patients presented with reactive lymphoid hyperplasia (7/10), 6/10 showed granulomatous inflammation. All cases showed some normal GCs but in 9/10 these concurred to a varying degree with hyperplastic, ill-defined GCs in the same LN. The percentage of ill-defined GCs correlated significantly with the percentage of circulating CD21low B cells suggesting a common origin of both immune reactions. In 9/10 CVID LNs significantly higher numbers of infiltrating CD8+ T cells were found in GCs of CVID patients compared to controls, but no HHV-8 and only in 2/10 LNs EBV infection was detected. Class switched plasma cells (PCs) were severely reduced in 8/10 LNs and if present, rarely found in the medulla of the LN. Based on the presence of large GCs in all examined patients, the reduction of circulating memory B cells and PCs points towards a failure of GC output rather than GC formation in CVID patients with lymphadenopathy.

Omenn syndrome associated with a functional reversion due to a somatic second-site mutation in CARD11 deficiency

Omenn syndrome (OS) is a severe immunodeficiency associated with erythroderma, lymphoproliferation, elevated IgE, and hyperactive oligoclonal T cells. A restricted T-cell repertoire caused by defective thymic T-cell development and selection, lymphopenia with homeostatic proliferation, and lack of regulatory T cells are considered key factors in OS pathogenesis. We report 2 siblings presenting with cytomegalovirus (CMV) and Pneumocystis jirovecii infections and recurrent sepsis; one developed all clinical features of OS. Both carried homozygous germline mutations in CARD11 (p.Cys150*), impairing NF-κB signaling and IL-2 production. A somatic second-site mutation reverting the stop codon to a missense mutation (p.Cys150Leu) was detected in tissue-infiltrating T cells of the OS patient. Expression of p.Cys150Leu in CARD11-deficient T cells largely reconstituted NF-κB signaling. The reversion likely occurred in a prethymic T-cell precursor, leading to a chimeric T-cell repertoire. We speculate that in our patient the functional advantage of the revertant T cells in the context of persistent CMV infection, combined with lack of regulatory T cells, may have been sufficient to favor OS. This first observation of OS in a patient with a T-cell activation defect suggests that severely defective T-cell development or homeostatic proliferation in a lymphopenic environment are not required for this severe immunopathology.

MicroRNA-146a reduces MHC-II expression via targeting JAK/STAT signaling in dendritic cells after stem cell transplantation.

Acute Graft-versus-host disease (GVHD) is a major immunological complication after allogeneic hematopoietic cell transplantation and a better understanding of the molecular regulation of the disease could help to develop novel targeted therapies. Here we found that a G/C polymorphism within the human microRNA-146a (miR-146a) gene of transplant recipients, which causes reduced miR-146a levels, was strongly associated with the risk of developing severe acute GVHD (n=289). 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.

Isotype-switched follicular lymphoma displays dissociation between activation-induced cytidine deaminase expression and somatic hypermutation

In B-cells, activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin genes. AID introduces mutations in immunoglobulin variable regions (IGV) during B-cell receptor affinity maturation, but may also introduce aberrant mutations into non-immunoglobulin genes, most commonly BCL6. Follicular lymphoma (FL) B-cells constitutively express AID and undergo CSR, SHM and aberrant SHM. We have studied AID expression, the presence of SHM mutations, CSR, and aberrant SHM in BCL6 in a cohort of 75 FL patients. Whereas IgM-expressing (non-switched) FL were characterized by an expected positive correlation between AID and IGV and BCL6 mutations, isotype-switched FL showed dissociation between AID expression and aberrant SHM, and inverse correlation between SHM and AID expression. Our results unveil two manifest biological subgroups of FL and indicate that the specific dissociation between AID and SHM after isotype switch may correlate with the clinical outcome of this heterogeneous disease.