Anja K. Wege

Humanized tumor mice—A new model to study and manipulate the immune response in advanced cancer therapy

The immunological impact on antibody‐based anticancer therapies remains incompletely understood due to the lack of appropriate animal models for in vivo analysis. Here, we present a novel humanized tumor mouse (HTM) model, generated by concurrent transplantation of human hematopoietic stem cells (HSCs) and human breast cancer cells in neonatal NOD‐scid IL2Rγnull mice. Five weeks after intrahepatic transplantation, a functional human immune system was developed in all organs, and, in addition, tumor cells were detectable in lung and bone marrow (early dissemination). After 3 months posttransplant, tumor‐cell effusions and macroscopic tumors associated with liver or spleen were found. Furthermore, disseminated cells in different lymphoid and nonlymphoid organs were measurable. Tumor growth was accompanied by specific T‐cell maturation and tumor cell‐specific T‐cell activation. In addition, Natural–Killer cell accumulation and activation were observed in HTM, which was further enhanced upon IL‐15 treatment facilitating the possibility of immune cell modulation in, e.g., antibody‐dependent cellular cytotoxicity‐based immunotherapeutic approaches. This novel mouse model makes it possible to combine transfer of MHC mismatched tumor cells together with human HSCs resulting in a solid coexistence and interaction without evidence for rejection. Overall, humanized tumor mice represent a powerful in vivo model that for the first time permits the investigation of human immune system‐related target cancer therapy and resistance.

Generation and characterization of alpha‐chymase‐Cre transgenic mice

The Cre‐loxP technology allows the introduction of somatic gene alterations in a tissue and/or cell type specific manner. The development of transgenes that target Cre expression to specific cell types is a critical component in this system. Here, we describe the generation and characterization of transgenic mouse lines expressing Cre recombinase under the control of the baboon α‐chymase promoter, designated Chm:Cre, in order to direct Cre expression specifically to mouse mast cells. Chm:Cre expression was detected in mast cells in lung and colon tissue. Cre‐mediated recombination in these mice identified a population of mature tissue resident mast cells using ROSA26R reporter mice. No Cre‐expression and Cre‐mediated recombination was induced in in vitro generated bone marrow derived mast cells or mast cells isolated from the peritoneal cavity indicating that Cre‐expression under the control of the α‐chymase promoter is solely activated in tissue resident mast cells. These Chm:Cre transgenic mice represent a useful tool to specifically inactivate genes of interest in mast cells of these tissues. genesis 46:163–166, 2008.

Humanized Mice, a New Model To Study the Influence of Drug Treatment on Neonatal Sepsis

ABSTRACT Bacterial infection with group B Streptococcus (GBS) represents a prominent threat to neonates and fetuses in the Western world, causing severe organ damage and even death. To improve current therapeutic strategies and to investigate new approaches, an appropriate in vivo model to study the immune response of a human immune system is needed. Therefore, we introduced humanized mice as a new model for GBS-induced sepsis. Humanized mice feature deficiencies similar to those found in neonates, such as lower immunoglobulin levels and myeloid cell dysfunction. Due to the husbandry in specific-pathogen-free (SPF) facilities, the human immune cells in these mice also exhibit a naive phenotype which mimics the conditions in fetuses/neonates. Following infection, cytokine release and leukocyte trafficking from the bone marrow to the lymphoid organ (spleen) and into the peritoneum (site of infection) as well as bacterial spreading and clearance were traceable in the humanized mice. Furthermore, we investigated the effects of betamethasone and indomethacin treatment using this novel sepsis model. Although both drugs are commonly used in perinatal care, little is known about their effects on the neonatal immune system. Treatment of infected humanized mice not only induced the reduction of human leukocytes in the spleen but also increased the bacterial load in all analyzed organs, including the brain, which did not show infiltration of live GBS in untreated controls. These studies demonstrate the utility of the humanized mice as a new model to study an immature human immune response during bacterial infection and allow the investigation of side effects induced by various treatments.

Leishmania major Infection in Humanized Mice Induces Systemic Infection and Provokes a Nonprotective Human Immune Response

Background Leishmania (L.) species are the causative agent of leishmaniasis. Due to the lack of efficient vaccine candidates, drug therapies are the only option to deal with cutaneous leishmaniasis. Unfortunately, chemotherapeutic interventions show high toxicity in addition to an increased risk of dissemination of drug-resistant parasites. An appropriate laboratory animal based model is still missing which allows testing of new drug strategies in the context of human immune cells in vivo. Methodology/Principal Findings Humanized mice were infected subcutaneously with stationary phase promastigote L. major into the footpad. The human immune response against the pathogen and the parasite host interactions were analyzed. In addition we proved the versatility of this new model to conduct drug research studies by the inclusion of orally given Miltefosine. We show that inflammatory human macrophages get infected with Leishmania parasites at the site of infection. Furthermore, a Leishmania-specific human-derived T cell response is initiated. However, the human immune system is not able to prevent systemic infection. Thus, we treated the mice with Miltefosine to reduce the parasitic load. Notably, this chemotherapy resulted in a reduction of the parasite load in distinct organs. Comparable to some Miltefosine treated patients, humanized mice developed severe side effects, which are not detectable in the classical murine model of experimental leishmaniasis. Conclusions/Significance This study describes for the first time L. major infection in humanized mice, characterizes the disease development, the induction of human adaptive and innate immune response including cytokine production and the efficiency of Miltefosine treatment in these animals. In summary, humanized mice might be beneficial for future preclinical chemotherapeutic studies in systemic (visceral) leishmaniasis allowing the investigation of human immune response, side effects of the drug due to cytokine production of activated humane immune cells and the efficiency of the treatment to eliminate also not replicating (“hiding”) parasites.

IRAK-M Expression in Tumor Cells Supports Colorectal Cancer Progression through Reduction of Antimicrobial Defense and Stabilization of STAT3

Colorectal cancer (CRC) is associated with loss of epithelial barrier integrity, which facilitates the interaction of the immunological microenvironment with the luminal microbiome, eliciting tumor-supportive inflammation. An important regulator of intestinal inflammatory responses is IRAK-M, a negative regulator of TLR signaling. Here we investigate the compartment-specific impact of IRAK-M on colorectal carcinogenesis using a mouse model. We demonstrate that IRAK-M is expressed in tumor cells due to combined TLR and Wnt activation. Tumor cell-intrinsic IRAK-M is responsible for regulation of microbial colonization of tumors and STAT3 protein stability in tumor cells, leading to tumor cell proliferation. IRAK-M expression in human CRCs is associated with poor prognosis. These results suggest that IRAK-M may be a potential therapeutic target for CRC treatment.

Laser Ablation–Inductively Coupled Plasma Mass Spectrometry: An Emerging Technology for Detecting Rare Cells in Tissue Sections

Administering immunoregulatory cells to patients as medicinal agents is a potentially revolutionary approach to the treatment of immunologically mediated diseases. Presently, there are no satisfactory, clinically applicable methods of tracking human cells in patients with adequate spatial resolution and target cell specificity over a sufficient period of time. Laser ablation–inductively coupled plasma mass spectrometry (LA-ICP-MS) represents a potential solution to the problem of detecting very rare cells in tissues. In this article, this exquisitely sensitive technique is applied to the tracking of gold-labeled human regulatory macrophages (Mregs) in immunodeficient mice. Optimal conditions for labeling Mregs with 50-nm gold particles were investigated by exposing Mregs in culture to variable concentrations of label: Mregs incubated with 3.5 × 109 particles/ml for 1 h incorporated an average of 3.39 × 108 Au atoms/cell without loss of cell viability. Analysis of single, gold-labeled Mregs by LA-ICP-MS registered an average of 1.9 × 105 counts/cell. Under these conditions, 100% labeling efficiency was achieved, and label was retained by Mregs for ≥36 h. Gold-labeled Mregs adhered to glass surfaces; after 24 h of culture, it was possible to colabel these cells with human-specific 154Sm-tagged anti–HLA-DR or 174Yb-tagged anti-CD45 mAbs. Following injection into immunodeficient mice, signals from gold-labeled human Mregs could be detected in mouse lung, liver, and spleen for at least 7 d by solution-based inductively coupled plasma mass spectrometry and LA-ICP-MS. These promising results indicate that LA-ICP-MS tissue imaging has great potential as an analytical technique in immunology.

Myeloid‐derived suppressor cell functionality and interaction with Leishmania major parasites differ in C57BL/6 and BALB/c mice

Myeloid‐derived suppressor cells (MDSCs) represent a heterogeneous population of CD11b+ cells. According to the surface molecules Ly6G and Ly6C (where Ly6G and Ly6C are lymphocyte antigen 6, locus G and C, respectively), MDSCs are further divided into monocytic (Mo‐MDSCs, CD11b+/Ly6Chigh/Ly6G−) and polymorphonucleated suppressor cells (PMN‐MDSCs, CD11b+/Ly6Cint/Ly6G+). Most published manuscripts focus on the suppressive role of MDSCs in cancer, whereas their impact on adaptive immunity against obligatory intracellular parasites is not well understood. Furthermore, it is not clear how the genetic background of mice influences MDSC functionality. Therefore, we implemented an experimental model of leishmaniasis, and analyzed MDSC maturation and the impact of MDSCs on the parasite‐specific T‐cell responses in resistant C57BL/6 and susceptible BALB/c mice. This experimental setup demonstrated the impaired ability of BALB/c mice to produce Mo‐MDSCs when compared with C57BL/6 mice. This phenotype is detectable after subcutaneous infection with parasites and is specifically represented by a reduced accumulation of Mo‐MDSCs at the site of infection in BALB/c mice. Moreover, infected C57BL/6‐derived MDSCs were able to suppress Leishmania‐specific CD4+ T‐cell proliferation, whereas BALB/c‐derived MDSCs harboring parasites lost this suppressive function. In conclusion, we demonstrate that (i) genetic background defines MDSC differentiation; and (ii) Leishmania major parasites are able to modulate the suppressive effect of MDSCs in a strain‐dependent manner.

Neutralization of LIGHT ameliorates acute dextran sodium sulphate-induced intestinal inflammation

Emerging data indicate that alterations in the expression of tumour necrosis factor (TNF) superfamily members play a crucial role in the pathogenesis of intestinal inflammation. Recent results demonstrated that sustained transgenic expression of lymphotoxin‐like inducible protein that competes with glycoprotein D for binding herpesvirus entry mediator on T cells (LIGHT; TNFSF14) induced severe intestinal inflammation, suggesting a specific role of LIGHT‐mediated signalling to the intestinal compartment. In order to dissect the role of LIGHT in intestinal inflammation, we used LIGHT‐deficient mice in the mouse model of acute dextran sodium sulphate‐induced colitis. Interestingly, LIGHT‐deficient mice were characterized by strongly reduced signs of intestinal inflammation compared with wild‐type mice in this experimental model. Determination of mouse LIGHT mRNA expression in colon tissues of wild‐type mice revealed a strong induction of mouse LIGHT mRNA expression during acute DSS‐induced colitis. We therefore generated anti‐mouse LIGHT monoclonal antibodies in LIGHT‐deficient mice which bind specifically to LIGHT and are capable of neutralizing the activity of LIGHT in vitro and in vivo. With these antibodies, we demonstrated that neutralization of LIGHT during acute DSS‐induced colitis resulted in reduced signs of intestinal inflammation. These data suggest that LIGHT is an important mediator in intestinal inflammation and may serve as a new target for therapeutic intervention.

Co-transplantation of human hematopoietic stem cells and human breast cancer cells in NSG mice: A novel approach to generate tumor cell specific human antibodies

Humanized tumor mice (HTM) were generated by the co-transplantation of human hematopoietic stem cells and human breast cancer cells overexpressing HER2 into neonatal NOD-scid IL2Rγnull (NSG) mice. These mice are characterized by the development of a human immune system in combination with human breast cancer growth. Due to concurrent transplantation into newborn mice, transfer of MHC-mismatched tumor cells resulted in solid coexistence and immune cell activation (CD4+ T cells, natural killer cells, and myeloid cells), but without evidence for rejection. Histological staining of the spleen of HTM revealed co-localization of human antigen-presenting cells together with human T and B cells allowing MHC-dependent interaction, and thereby the generation of T cell-dependent antibody production. Here, we investigated the capability of these mice to generate human tumor-specific antibodies and correlated immunoglobulin titers with tumor outgrowth. We found detectable IgM and also IgG amounts in the serum of HTM, which apparently controlled tumor development when IgG serum concentrations were above 10 µg/ml. Western blot analyses revealed that the tumor-specific antibodies generated in HTM did not recognize HER2/neu antigens, but different, possibly relevant antigens for breast cancer therapy. In conclusion, HTM offer a novel approach to generate complete human monoclonal antibodies that do not require further genetic manipulation (e. g., humanization) for a potential application in humans. In addition, efficacy and safety of the generated antibodies can be tested in the same mouse model under human-like conditions. This might be of particular interest for cancer subtypes with no currently available antibody therapy.

Characteristics of “Tip-DCs and MDSCs” and Their Potential Role in Leishmaniasis

Since the first description of dendritic cells (DCs) by Steinman and Cohn (1973), the myeloid lineage of leukocytes was investigated intensively. Nowadays it is obvious that myeloid cells, especially DCs, are crucial for the adaptive and innate immune response against intracellular pathogens such as Leishmania major parasites. Based on the overlapping expression of molecules that were commonly used to classify myeloid cells, it becomes difficult to denominate those cell types precisely. Of note, most of these markers used for myeloid cell identification are expressed on a broad range of myeloid cells, and should therefore be handled with care if used for subtyping of myeloid cells. In this mini-review we aim to discuss the relative impact of DCs that release TNF and nitric oxide (Tip-DCs) and myeloid cells with suppressive capacities (myeloid-derived suppressor cells, MDSCs) in infectious diseases such as experimental leishmaniasis. In our point of view it cannot be excluded that the novel subsets that were denominated as “Tip-DCs” and “MDSCs” might not be classical “subsets” but rather represent myeloid cells in a transient maturation stage expressing different genes, in response to the surrounding environment.