Ursula R. Sorg

Enforced viral replication activates adaptive immunity and is essential for the control of a cytopathic virus.

The innate immune system limits viral replication via type I interferon and also induces the presentation of viral antigens to cells of the adaptive immune response. Using infection of mice with vesicular stomatitis virus, we analyzed how the innate immune system inhibits viral propagation but still allows the presentation of antigen to cells of the adaptive immune response. We found that expression of the gene encoding the inhibitory protein Usp18 in metallophilic macrophages led to lower type I interferon responsiveness, thereby allowing locally restricted replication of virus. This was essential for the induction of adaptive antiviral immune responses and, therefore, for preventing the fatal outcome of infection. In conclusion, we found that enforced viral replication in marginal zone macrophages was an immunological mechanism that ensured the production of sufficient antigen for effective activation of the adaptive immune response.

A Novel Nonobese Diabetic/Severe Combined Immunodeficient Xenograft Model for Chronic Lymphocytic Leukemia Reflects Important Clinical Characteristics of the Disease

We here describe a novel xenograft model of chronic lymphocytic leukemia (CLL) generated by infusion of human primary CLL cells into immunodeficient nonobese/severe combined immunodeficient (NOD/SCID) mice. Combined i.v. and i.p. injection of peripheral blood mononuclear cells (PBMC) from 39 patients with CLL resulted in highly reproducible splenic (37 of 39) and peritoneal (35 of 39) engraftment, which remained stable over a time span of 4 to 8 weeks. By comparison, recovery of leukemic cells from bone marrow (21 of 39) or peripheral blood (8 of 22) was substantially lower. The engraftment pattern of CLL PBMC 4 weeks posttransplant was correlated with clinical disease activity: infusion of PBMC from donors with Binet stage A, lymphocyte doubling time of >12 months, and normal lactate dehydrogenase (LDH) serum levels led to marked engraftment of T cells whereas comparably few tumor cells could be detected. In contrast, NOD/SCID mice receiving PBMC from donors with advanced stage Binet C, lymphocyte doubling time of <12 months, and elevated LDH serum levels exhibited predominant engraftment of tumor cells and comparably low numbers of T cells. These results suggest that this model reflects the heterogeneity and important clinical characteristics of the disease, and thus may serve as a tool for preclinical drug testing and investigation of the pathophysiology of CLL.

Stringent Regulation of DNA Repair During Human Hematopoietic Differentiation: A Gene Expression and Functional Analysis

For the lymphohematopoietic system, maturation‐dependent alterations in DNA repair function have been demonstrated. Because little information is available on the regulatory mechanisms underlying these changes, we have correlated the expression of DNA damage response genes and the functional repair capacity of cells at distinct stages of human hematopoietic differentiation. Comparing fractions of mature (CD34−), progenitor (CD34+38+), and stem cells (CD34+38−) isolated from umbilical cord blood, we observed: 1) stringently regulated differentiation‐dependent shifts in both the cellular processing of DNA lesions and the expression profiles of related genes and 2) considerable interindividual variability of DNA repair at transcriptional and functional levels. The respective repair phenotype was found to be constitutively regulated and not dominated by adaptive response to acute DNA damage. During blood cell development, the removal of DNA adducts, the resealing of repair gaps, the resistance to DNA‐reactive drugs clearly increased in stem or mature compared with progenitor cells of the same individual. On the other hand, the vast majority of differentially expressed repair genes was consistently upregulated in the progenitor fraction. A positive correlation of repair function and transcript levels was found for a small number of genes such as RAD23 or ATM, which may serve as key regulators for DNA damage processing via specific pathways. These data indicate that the organism might aim to protect the small number of valuable slow dividing stem cells by extensive DNA repair, whereas fast‐proliferating progenitor cells, once damaged, are rather eliminated by apoptosis.

Protection of hematopoietic cells from O6‐alkylation damage by O6‐methylguanine DNA methyltransferase gene transfer: studies with different O6‐alkylating agents and retroviral backbones

Abstract: Overexpression of O6‐methylguanine DNA methyltransferase (MGMT) can protect hematopoietic cells from O6‐alkylation damage. To identify possible clinical applications of this technology we compared the effect of MGMT gene transfer on the hematotoxicity induced by different O6‐alkylating agents in clinical use: the chloroethylnitrosoureas ACNU, BCNU, CCNU and the tetrazine derivative temozolomide. In addition, various retroviral vectors expressing the MGMT‐cDNA were investigated to identify optimal viral backbones for hematoprotection by MGMT expression. Protection from ACNU, BCNU, CCNU or temozolomide toxicity was evaluated utilizing a Moloney murine leukemia virus‐based retroviral vector (N2/Zip‐PGK‐MGMT) to transduce primary murine bone marrow cells. Increased resistance in murine colony‐forming units (CFU) was demonstrated for all four drugs. In comparison to mock‐transduced controls, after transduction with N2/Zip‐PGK‐MGMT the IC50 for CFU increased on average 4.7‐fold for ACNU, 2.5‐fold for BCNU, 6.3‐fold for CCNU and 1.5‐fold for temozolomide. To study the effect of the retroviral backbone on hematoprotection various vectors expressing the human MGMT‐cDNA from a murine embryonic sarcoma virus LTR (MSCV‐MGMT) or a hybrid spleen focus‐forming/murine embryonic sarcoma virus LTR (SF1‐MGMT) were compared with the N2/Zip‐PGK‐MGMT vector. While all vectors increased resistance of transduced human CFU to ACNU, the SF1‐MGMT construct was most efficient especially at high ACNU concentrations (8–12 µg/ml). Similar results were obtained for protection of murine high‐proliferative‐potential colony‐forming cells. These data may help to optimize treatment design and retroviral constructs in future clinical studies aiming at hematoprotection by MGMT gene transfer.

Clonal inventory screens uncover monoclonality following serial transplantation of MGMTP140K-transduced stem cells and dose-intense chemotherapy

Gene transfer of mutant O(6)-methylguanine-DNA-methyltransferase (MGMT(P140K)) into hematopoietic stem cells (HSCs) protects hematopoiesis from alkylating agents and allows efficient in vivo selection of transduced HSCs. However, insertional mutagenesis, high regenerative stress associated with selection, and the genotoxic potential of alkylating drugs represent considerable risk factors for clinical applications of this approach. Therefore, we investigated the long-term effect of MGMT(P140K) gene transfer followed by repetitive, dose-intensive treatment with alkylating agents in a murine serial bone marrow transplant model and assessed clonality of hematopoiesis up to tertiary recipients. The substantial selection pressure resulted in almost completely transduced hematopoiesis in all cohorts. Ligation-mediated PCR and next-generation sequencing identified several repopulating clones carrying vector insertions in distinct genomic regions that were ∼ 9 kb of size (common integration sites). Beside polyclonal reconstitution in the majority of the mice, we also detected monoclonal or oligoclonal repopulation patterns with HSC clones showing vector insertions in the Usp10 or Tubb3 gene. Interestingly, neither Usp10, Tubb3, nor any of the genes located in common integration sites have been linked to clonal expansion in previous preclinical or clinical gene therapy trials. However, a considerable number of these genes are involved in DNA damage response and cell fate decision pathways following cytostatic drug application. Thus, in summary, our study advocates ligation-mediated PCR and next generation sequencing as an effective and reliable method to identify gene products associated with clonal survival in specific experimental settings such as chemoselection using alkylating agents.

One-step molecular HLA-DR prescreening employing a set of 14 sequence specific oligonucleotides in a non-radioactive tetramethylammonium chloride hybridization protocol.

A novel non‐radioactive protocol for molecular generic HLA‐DR typing is introduced, employing sequence specific oligonucleotides (SSOs) enzymatically 3′‐labelled with biotin‐14‐dATP via terminal deoxynucleotidyltransferase in a tetramethylammonium chloride hybridization procedure. The detection reaction is carried out, using streptavidine conjugated horseradish peroxidase which is bound to the SSOs, in combination with a light emitting detection system. Fourteen SSOs and one control SSO are employed for generic HLA‐DR typing in a one‐step protocol. In order to demonstrate the suitability of this procedure, 5 homozygous typing cell lines and samples of 11 pretyped individuals which include most serologically defined HLA‐DR specificities (DR1, 2, 3, 4, 11, 12, 6, 7, 8, 9,10, 52, and DR53) are analysed with the panel of 14 SSOs. The typing results show that this protocol, which avoids the use of radioisotopes, combines high specificity and easy handling. It also allows typing of poorly amplified samples because even after longer exposition times no false positive signals were observed and is particularly suitable for routine molecular HLA‐DR typing on the generic level. In addition it can easily be adapted to DP and DQ typing or DR subtyping.

Cooperative role of lymphotoxin β receptor and tumor necrosis factor receptor p55 in murine liver regeneration

BACKGROUND & AIMS The liver exhibits a unique capacity for regeneration in response to injury. Lymphotoxin-β receptor (LTβR), a core member of the tumor necrosis factor (TNF)/tumor necrosis factor receptor (TNFR) superfamily is known to play an important role in this process. However, the function of LTβR during pathophysiological alterations and its molecular mechanisms during liver regeneration are so far ill-characterized. METHODS LTβR(-/-) mice were subjected to 70% hepatectomy and liver regeneration capacity, bile acid profiles, and transcriptome analysis were performed. RESULTS LTβR(-/-) deficient mice suffered from increased and prolonged liver tissue damage after 70% hepatectomy, accompanied by deregulated bile acid homeostasis. Pronounced differences in the expression patterns of genes relevant for bile acid synthesis and recirculation were observed. LTβR and TNFRp55 share downstream signalling elements. Therefore, LTβR(-/-) mice were treated with etanercept to create mice functionally deficient in both signalling pathways. Strikingly, the combined blockade of TNFRp55 and LTβR signalling leads to complete failure of liver regeneration resulting in death within 24 to 48h after PHx. Transcriptome analysis revealed a marked disparity in gene expression programs in livers of LTβR(-/-) and etanercept-treated LTβR(-/-) vs. wild-type animals after PHx. Murinoglobulin 2 was identified as a significantly differentially regulated gene. CONCLUSIONS LTβR is essential for efficient liver regeneration and cooperates with TNFRp55 in this process. Differences in survival kinetics strongly suggest distinct functions for these two cytokine receptors in liver regeneration. Failure of TNFR and LTβR signalling renders liver regeneration impossible.

The Lymphotoxin β Receptor Is Essential for Upregulation of IFN-Induced Guanylate-Binding Proteins and Survival after Toxoplasma gondii Infection

Lymphotoxin β receptor (LTβR) signaling plays an important role in efficient initiation of host responses to a variety of pathogens, encompassing viruses, bacteria, and protozoans via induction of the type I interferon response. The present study reveals that after Toxoplasma gondii infection, LTβR−/− mice show a substantially reduced survival rate when compared to wild-type mice. LTβR−/− mice exhibit an increased parasite load and a more pronounced organ pathology. Also, a delayed increase of serum IL-12p40 and a failure of the protective IFNγ response in LTβR−/− mice were observed. Serum NO levels in LTβR−/− animals rose later and were markedly decreased compared to wild-type animals. At the transcriptional level, LTβR−/− animals exhibited a deregulated expression profile of several cytokines known to play a role in activation of innate immunity in T. gondii infection. Importantly, expression of the IFNγ-regulated murine guanylate-binding protein (mGBP) genes was virtually absent in the lungs of LTβR−/− mice. This demonstrates clearly that the LTβR is essential for the induction of a type II IFN-mediated immune response against T. gondii. The pronounced inability to effectively upregulate host defense effector molecules such as GBPs explains the high mortality rates of LTβR−/− animals after T. gondii infection.

The role of the lymphotoxin-β receptor (LTβR) in hepatocyte-mediated liver regeneration

Background The lymphotoxin beta receptor (LTbR) is a prototypic member of the TNF/TNFR superfamily and has diverse functions in regulating immune responses against pathogens, in organogenesis and maintenance of structural integrity of secondary lymphoid tissues [1]. Mice deficient in LTbR (LTbR) show impaired resistance to intracellular pathogens and defects in peripheral lymphoid tissues [2,3]. Importantly, LTbR mice also exhibit markedly reduced survival after partial (70%) hepatectomy (PHx) [4], compared to WT controls. Liver mass is tightly regulated to 5% of the body weight and the liver retains a remarkable capacity for regeneration in response to acute injury. Loss of at least 30% of liver mass leads to synchronized proliferation of normally quiescent mature hepatocytes (compensatory hyperplasia) until physiologic liver mass is regained. Recently, it has been shown that lymphotoxins required for liver regeneration are produced by T cells immigrating into the liver during the regeneration process [5]. During this regeneration process, it is mandatory that the liver continues to perform its essential functions, such as protein synthesis, glycogen storage and bile secretion. There is evidence, that nuclear receptordependent bile acid signaling is required to initiate efficient liver regeneration [6] and that signaling through the TNFRp55, another core member of the TNF/TNFR superfamily is an essential part of early liver regeneration [7]. Interestingly, signaling pathways through TNFRp55 and LTbR share downstream signaling components. However, the molecular mechanisms orchestrated by LTbR and TNFRp55 signaling remain incompletely defined. Results Our data confirms that following PHx LTbR mice have a decreased survival rate compared to WT mice (62 % vs. 90 %, respectively). Surviving LTbR mice show a rate of liver regeneration comparable to WT animals. Post PHx HE most prominent among these was a markedly decreased expression of murinoglobulin-2, a proteinase inhibitor of the a2-macroglobulin family, in LTbR mice.