Andreas Beilhack

Shifting foci of hematopoiesis during reconstitution from single stem cells

To reveal the early events and dynamics of hematopoietic reconstitution in living animals in real-time, we used bioluminescence imaging to monitor engraftment from single luciferase-labeled hematopoietic stem cells (HSC) in irradiated recipients. Transplanted HSC generated discrete foci in the spleen and bone marrow (BM), at a frequency that correlated with BM compartment size. Initially detected foci could expand locally, seed other sites in BM or spleen, and/or recede with different kinetics. These studies reveal dynamic and variable patterns of engraftment from highly purified HSC and indicate that the final overall contribution of individual HSC to hematopoietic chimerism does not depend on the specific site of initial engraftment and expansion.

In vivo trafficking and survival of cytokine-induced killer cells resulting in minimal GVHD with retention of antitumor activity.

Cytokine-induced killer (CIK) cells are ex vivo-expanded T lymphocytes expressing both natural killer (NK)- and T-cell markers. CIK cells are cytotoxic against autologous and allogeneic tumors. We previously showed that adoptive transfer of allogeneic CIK cells in a murine model caused minimal graft-versus-host disease (GVHD). However, the precise mechanism of reduced GVHD is not fully understood. Therefore, we evaluated the trafficking and survival of luciferase-expressing CIK cells in an allogeneic bone marrow transplant model. The initial trafficking patterns of CIK cells were similar to conventional T cells that induced GVHD; however, CIK cells infiltrated GVHD target tissues much less and transiently. CIK cells accumulated and persisted in tumor sites, resulting in tumor eradication. We evaluated different properties of CIK cells compared with conventional T cells, demonstrating a slower division rate of CIK cells, higher susceptibility to apoptosis, persistent increased expression of interferon gamma (IFN-gamma), and reduced acquisition of homing molecules required for entry of cells into inflamed GVHD target organs that lack expression of NKG2D ligands recognized by CIK cells. Due to these properties, allogeneic CIK cells had reduced expansion and caused less tissue damage. We conclude that CIK cells have the potential to separate graft-versus-tumor effects from GVHD.

Targeting Localized Immune Suppression Within the Tumor Through Repeat Cycles of Immune Cell-oncolytic Virus Combination Therapy

A major limitation to the use of immunotherapy in the treatment of cancer has been the localized immune suppressive environment within the tumor. Although there is evidence that tumor-selective (oncolytic) viruses may help to overcome this immune suppression, a primary limitation to their use has been limited systemic delivery potential, especially in the face of antiviral immunity. We recently demonstrated that tumor-trafficking immune cells can efficiently deliver oncolytic viral therapies to their tumor targets. These cells act as both a therapeutic agent and also a carrier vehicle for the oncolytic virus. Here, we demonstrate that such delivery is also possible in the face of pre-existing antiviral immunity, so overcoming the limited systemic delivery of naked, cell-free virus. It was also found that treatment of previously immunized mice or repeat treatments leading to immunization resulted in a switch from a primarily oncolytic to an immunotherapeutic mechanism of action. Furthermore, repeat cycles of treatment with combination immune cell-viral therapy resulted in increased tumor infiltration of effector T-cells and a general reduction in the levels of known immune suppressive lymphocyte populations. This therefore represents a novel and effective means to overcome localized immune suppression within the tumor microenvironment.

Generation of highly effective and stable murine alloreactive Treg cells by combined anti-CD4 mAb, TGF-β, and RA treatment.

The transfer of alloreactive regulatory T (aTreg) cells into transplant recipients represents an attractive treatment option to improve long‐term graft acceptance. We recently described a protocol for the generation of aTreg cells in mice using a nondepleting anti‐CD4 antibody (aCD4). Here, we investigated whether adding TGF‐β and retinoic acid (RA) or rapamycin (Rapa) can further improve aTreg‐cell generation and function. Murine CD4+ T cells were cultured with allogeneic B cells in the presence of aCD4 alone, aCD4+TGF‐β+RA or aCD4+Rapa. Addition of TGF‐β+RA or Rapa resulted in an increase of CD25+Foxp3+‐expressing T cells. Expression of CD40L and production of IFN‐γ and IL‐17 was abolished in aCD4+TGF‐β+RA aTreg cells. Additionally, aCD4+TGF‐β+RA aTreg cells showed the highest level of Helios and Neuropilin‐1 co‐expression. Although CD25+Foxp3+ cells from all culture conditions displayed complete demethylation of the Treg‐specific demethylated region, aCD4+TGF‐β+RA Treg cells showed the most stable Foxp3 expression upon restimulation. Consequently, aCD4+TGF‐β+RA aTreg cells suppressed effector T‐cell differentiation more effectively in comparison to aTreg cells harvested from all other cultures, and furthermore inhibited acute graft versus host disease and especially skin transplant rejection. Thus, addition of TGF‐β+RA seems to be superior over Rapa in stabilising the phenotype and functional capacity of aTreg cells.

MB3W1 is an orthotopic xenograft model for anaplastic medulloblastoma displaying cancer stem cell- and Group 3-properties.

BackgroundMedulloblastoma is the most common malignant brain tumor in children and can be divided in different molecular subgroups. Patients whose tumor is classified as a Group 3 tumor have a dismal prognosis. However only very few tumor models are available for this subgroup.MethodsWe established a robust orthotopic xenograft model with a cell line derived from the malignant pleural effusions of a child suffering from a Group 3 medulloblastoma.ResultsBesides classical characteristics of this tumor subgroup, the cells display cancer stem cell characteristics including neurosphere formation, multilineage differentiation, CD133/CD15 expression, high ALDH-activity and high tumorigenicity in immunocompromised mice with xenografts exactly recapitulating the original tumor architecture.ConclusionsThis model using unmanipulated, human medulloblastoma cells will enable translational research, specifically focused on Group 3 medulloblastoma.

Selective NFAT targeting in T cells ameliorates GvHD while maintaining antitumor activity

Significance Patients suffering from hematologic malignancies might have no alternative to eliminating their morbid immune system. Although lethal irradiation or chemotherapy largely eradicates tumor cells, it also destroys the hematopoietic system, which necessitates transplantation of bone marrow stem cells from a healthy donor. T cells within the graft attack remaining tumor cells. This graft-versus-leukemia (GvL) effect is highly appreciated, because it prevents tumor relapses. Coevally, however, T cells attack nonmalignant tissue of the host with life-threating consequences called graft-versus-host disease (GvHD). We found that absence of the transcription factors NFAT (nuclear factor of activated T cells) in T cells prevents harmful GvHD, but preserves the valuable GvL. Therefore, instead of broad immune suppression, we propose to target NFAT specifically during allogenic stem cell transplantation. Graft-versus-host disease (GvHD) is a life-threatening immunological complication after allogenic hematopoietic stem cell transplantation (allo-HCT). The intrinsic graft-versus-leukemia (GvL) effect, however, is the desirable curative benefit. Patients with acute GvHD are treated with cyclosporine A (CsA) or tacrolimus (FK506), which not only often causes severe adverse effects, but also interferes with the anticipated GvL. Both drugs inhibit calcineurin, thus at first suppressing activation of the nuclear factor of activated T cells (NFAT). Therefore, we explored the specific contribution of individual NFAT factors in donor T cells in animal models of GvHD and GvL. Ablation of NFAT1, NFAT2, or a combination of both resulted in ameliorated GvHD, due to reduced proliferation, target tissue homing, and impaired effector function of allogenic donor T cells. In contrast, the frequency of Foxp3+ regulatory T (Treg) cells was increased and NFAT-deficient Tregs were fully protective in GvHD. CD8+ T-cell recall response and, importantly, the beneficial antitumor activity were largely preserved in NFAT-deficient effector T cells. Thus, specific inhibition of NFAT opens an avenue for an advanced therapy of GvHD maintaining protective GvL.

Src-kinase inhibitors sensitize human cells of myeloid origin to Toll-like-receptor-induced interleukin 12 synthesis.

Src-kinase inhibitors hold great potential as targeted therapy against malignant cells. However, such inhibitors may also affect nonmalignant cells and cause pronounced off-target effects. We investigated the role of the dual kinase inhibitor dasatinib on human myeloid cells. Dasatinib is clinically used for the treatment of bcr/abl⁺ leukemias because it blocks the mutated tyrosine kinase abl. To understand its effect on the development of antigen-specific T-cell responses, we assessed antigen-specific priming of human, naïve T cells. In surprising contrast to the direct inhibition of T-cell activation by dasatinib, pretreatment of maturing dendritic cells (DCs) with dasatinib strongly enhanced their stimulatory activity. This effect strictly depended on the activating DC stimulus and led to enhanced interleukin 12 (IL-12) production and T-cell responses of higher functional avidity. Src-kinase inhibitors, and not conventional tyrosine kinase inhibitors, increased IL-12 production in several cell types of myeloid origin, such as monocytes and classical or nonclassical DCs. Interestingly, only human cells, but not mouse or macaques DCs, were affected. These data highlight the potential immunostimulatory capacity of a group of novel drugs, src-kinase inhibitors, thereby opening new opportunities for chemoimmunotherapy. These data also provide evidence for a regulatory role of src kinases in the activation of myeloid cells.

Developmental adaptations of trypanosome motility to the tsetse fly host environments unravel a multifaceted in vivo microswimmer system

The highly motile and versatile protozoan pathogen Trypanosoma brucei undergoes a complex life cycle in the tsetse fly. Here we introduce the host insect as an expedient model environment for microswimmer research, as it allows examination of microbial motion within a diversified, secluded and yet microscopically tractable space. During their week-long journey through the different microenvironments of the fly´s interior organs, the incessantly swimming trypanosomes cross various barriers and confined surroundings, with concurrently occurring major changes of parasite cell architecture. Multicolour light sheet fluorescence microscopy provided information about tsetse tissue topology with unprecedented resolution and allowed the first 3D analysis of the infection process. High-speed fluorescence microscopy illuminated the versatile behaviour of trypanosome developmental stages, ranging from solitary motion and near-wall swimming to collective motility in synchronised swarms and in confinement. We correlate the microenvironments and trypanosome morphologies to high-speed motility data, which paves the way for cross-disciplinary microswimmer research in a naturally evolved environment. DOI: http://dx.doi.org/10.7554/eLife.27656.001

Production of BMP4 by endothelial cells is crucial for endogenous thymic regeneration

BMP4 produced by endothelial cells promotes thymic regeneration after acute damage by activating FOXN1 and its downstream targets. Regeneration circuits in the thymus Chemotherapy and radiation treatments in cancer patients damage a number of tissues and organs, including the thymus. Prolonged thymic damage can lead to T cell deficiency and increase susceptibility to the development of opportunistic infections and malignancies. Here, Wertheimer et al. have examined thymic regeneration in mice after sublethal total body radiation and document a critical role for bone morphogenetic protein 4 (BMP4) signaling in thymic regeneration. They found endothelial cells to be a critical source of BMP4 and propose that BMP4 produced by endothelial cells induces the expression of the transcription factor FOXN1 in thymic epithelial cells to promote thymic regeneration. These studies should eventually facilitate the development of treatment regimens to promote immune competence in patients undergoing chemotherapy and radiation treatments. The thymus is not only extremely sensitive to damage but also has a remarkable ability to repair itself. However, the mechanisms underlying this endogenous regeneration remain poorly understood, and this capacity diminishes considerably with age. We show that thymic endothelial cells (ECs) comprise a critical pathway of regeneration via their production of bone morphogenetic protein 4 (BMP4). ECs increased their production of BMP4 after thymic damage, and abrogating BMP4 signaling or production by either pharmacologic or genetic inhibition impaired thymic repair. EC-derived BMP4 acted on thymic epithelial cells (TECs) to increase their expression of Foxn1, a key transcription factor involved in TEC development, maintenance, and regeneration, and its downstream targets such as Dll4, a key mediator of thymocyte development and regeneration. These studies demonstrate the importance of the BMP4 pathway in endogenous tissue regeneration and offer a potential clinical approach to enhance T cell immunity.

Targeting of the WT191-138 fragment to human dendritic cells improves leukemia-specific T-cell responses providing an alternative approach to WT1-based vaccination.

Due to its immunogenicity and overexpression concomitant with leukemia progression, Wilms tumor protein 1 (WT1) is of particular interest for immunotherapy of AML relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT). So far, WT1-specific T-cell responses have mainly been induced by vaccination with peptides presented by certain HLA alleles. However, this approach is still not widely applicable in clinical practice due to common limitations of HLA restriction. Dendritic cell (DC) vaccines electroporated with mRNA encoding full-length protein have also been tested for generating WT1-derived peptides for presentation to T-cells. Alternatively, an efficient and broad WT1 peptide presentation could be elicited by triggering receptor-mediated protein endocytosis of DCs. Therefore, we developed antibody fusion proteins consisting of an antibody specific for the DEC205 endocytic receptor on human DCs and various fragments of WT1 as DC-targeting recombinant WT1 vaccines (anti-hDEC205-WT1). Of all anti-hDEC205-WT1 fusion proteins designed for overcoming insufficient expression, anti-hDEC205-WT110–35, anti-hDEC205-WT191–138, anti-hDEC205-WT1223–273, and anti-hDEC205-WT1324–371 were identified in good yields. The anti-hDEC205-WT191–138 was capable of directly inducing ex vivo T-cell responses by co-incubation of the fusion protein-loaded monocyte-derived mature DCs and autologous T-cells of either healthy or HSCT individuals. Furthermore, the DC-targeted WT191–138-induced specific T-cells showed a strong cytotoxic activity by lysing WT1-overexpressing THP-1 leukemia cells in vitro while sparing WT1-negative hematopoietic cells. In conclusion, our approach identifies four WT1 peptide-antibody fusion proteins with sufficient production and introduces an alternative vaccine that could be easily translated into clinical practice to improve WT1-directed antileukemia immune responses after allo-HSCT.