Table of Contents

Aims

Investigation of immunosuppression mechanisms and development of innovative immunotherapeutic strategies for human malignant melanoma using ret transgenic mouse melanoma model.

Background

An establishment of reliable mouse models of skin melanoma that resemble human melanoma is highly desirable. Conventional animal melanoma models (e.g. B16) are based on the tumor cell transplantation, in which the natural history of the disease tumor-host interactions are not comparable with the clinical situation. In contrast to B16 melanoma, recently developed transgenic mouse spontaneous melanoma models resemble human melanoma with respect to etiology, tumor genetics, histopathology and clinical development. We use one of these models, in which ret transgene is expressed in melanocytes under the control of metallothionein-I promoter (Ret-tg mice). After a short latency (1-3 months), around 25% mice develop skin melanoma metastasizing to lymph nodes, lungs, liver and brain. Primary tumors expressed melanoma associated antigens (MAA) tyrosinase, tyrosinase related protein (TRP)-1, TRP-2 and gp100. Ret-tg mice without macroscopic tumors could mount both antigen-unspecific (stimulation with Con A or with CD3/CD28 antibodies) and antigen-specific (ovalbumin or TRP-2 derived peptide) T-cell reactions, which did not differ from those in non-transgenic littermates. Thus, this mouse model of spontaneous skin melanoma allows testing new immunotherapeutic approaches at different stages of disease.

Projects

The following projects are currently running in our group. The references in brackets refer to the publication list (see left side, publications).

Project 1: Immunotherapy with activated memory T cells (MTC) specific for MAA

MAA-specific MTC could be a good source for generating therapeutic effector cells since they show higher frequency and react to antigens faster and stronger than naïve T cells. Our studies revealed that the bone marrow (BM) is a major site for persistence of tumor-specific memory T cells (1, 2, 4, 5, 12). Re-stimulation of MTC in vitro by dendritic cells (DC) loaded with tumor antigens led to IFN-γ production and the acquisition of antitumor cytotoxicity in vitro and in vivo (1, 4, 8, 11). Naive BM T cells could be also primed by resident DC resulting in generation of cytotoxic T cells, protective anti-tumor immunity and immunological memory (9, 10). Using ret transgenic melanoma model, we showed that we have shown that not only ret transgenic mice with macroscopic primary tumors but also ret transgenic mice without them contained BM MAA-specific CD8+ MTC with high activity in vitro and in vivo (15). This implies that the development and maintenance of functionally active melanoma-reactive MTC already at the preclinical stage of melanoma progression. In this project, we plan to develop an innovative adoptive cellular immunotherapy of human malignant melanoma with activated MAA-specific MTC. We will study effects of various protocols of immunotherapy with MAA-reactive MTC re-stimulated by DC loaded with melanoma antigens on melanoma growth and on T-cell mediated immune responses in transgenic mice. In addition, this therapy will be combined with the neutralization of immunosuppressive microenvironment (see also project 4).
Within this topic we have a small project to be done within a medical thesis or master thesis. If you are interested in this project, please use the contact form of Viktor Umansky.

Project 2: Interaction of dormant (stem) tumor cells and melanoma-specific memory T cells

Many solid tumors (including melanoma) appear to have a small population of stem cells that are resistant to chemotherapy and can perpetuate themselves indefinitely. These cancer stem cells thus far have been isolated from breast and brain tumors as well as blood. The exact origin of these cancer stem cells remains to be defined. Our studies in breast cancer patients and in melanoma bearing ret transgenic mice revealed the presence of small numbers of dormant tumor cells in the bone marrow (2, 15). Interestingly, dormant melanoma cells were also found in the bone marrow and lymph nodes of transgenic mice without macroscopic skin tumors (15). This correlated with the detection of functionally active melanoma-specific memory T cells in these organs. We aim to investigate whether these dormant melanoma cells possess the markers and functional properties of stem cells. In addition, their role in the maintenance of long-term survival and functional activity is under study.

Project 3: Characterization of regulatory T cells during melanoma progression

Although melanoma is immunogenic, melanoma cells escape the immune response and progress to metastatic disease due to the different mechanisms of immunosuppression, which include, in particular, CD4+CD25+FoxP3+ regulatory T cells (Treg). Recent studies reported an increased frequency of Treg in cancer patients. In mouse tumor transplantation models (in particular, in B16 melanoma), removal of CD4+CD25+ T cells enhanced antitumor immune responses suggesting that these cells can inhibit tumor-specific immune reactions. In Ret-tg mouse melanoma model, we found an accumulation of Treg in skin tumors and metastatic lymph nodes at early stages of melanoma progression that inversely correlated with Treg amounts in the bone marrow. Although anti-CD25 mAb injections resulted in the Treg depletion from lymphoid organs of transgenic mice, melanoma development was not significantly delayed. Furthermore, the treatment of mice with macroscopic tumors also failed to inhibit tumor progression, which correlated with the inability to deplete intratumoral Treg. By crossing ret transgenic mice with transgenic FoxP3-GDL mice that co-express eGFP, diphtheria toxin receptor (DTR) and luciferase under the control of Treg-specific FoxP3 promoter, we plan to study the therapeutic effect of specific Treg elimination in all lymphoid organs and tumors at different stages of melanoma progression.
Within this topic we have a small project to be done within a medical thesis or master thesis. If you are interested in this project, please use the contact form of Viktor Umansky.

Project 4: Neutralization of melanoma immunosuppressive microenvironment by inhibiting of myeloid derived suppressor cells

Sustained T cell activation and generation of chronic inflammatory immune responses are known to lead to the profound down-regulation of zeta-chain expression and the impairment of T cell activity. This impairment may be generated by Gr1+CD11b+ myeloid derived suppressor cells (MDSC) that are accumulated at the tumor site and secrete immunosuppressive factors (e.g., nitric oxide or TGF-beta). MDSC infiltrate progressing primary melanomas in Ret-tg mice that correlates with the down regulation of zeta-chain expression in tumor infiltrating T cells. Moreover, MDSC isolated from tumor bearing mice showed a strong suppression of T-cell proliferation in vitro. We aim to apply inhibitors of MDSC suppressive activity (such as phosphodiesterase-5 inhibitor, Viagra) preventing them to produce immunosuppressive molecules (e.g., nitric oxide or TGF-beta). This approach will permit to design innovative strategies for melanoma immunotherapy, in which inhibitors of the MDSC function (like Viagra) can be used alone or combined with other adoptive transfer of specifically re-stimulated MAA-specific memory T cells (see also project 1).

Project 5: Tolerogenic dendritic cells in Ret-transgenic mice during spontaneous melanoma development

DC are known to have a unique capacity for inducing primary T-cell-mediated immune responses against various tumor-associated antigens. This makes them attractive for antitumor immunotherapy. However, some DC can induce and maintain immune tolerance, while others are immunogenic. Immature or semi-mature DC are known to present antigens to T cells in the absence of appropriate co-stimulation that lead to T cell tolerance, deletion or to the development of regulatory T cells. These DC could be responsible for inhibition of effective anti-tumor T cell immune responses in vivo. During melanoma progression in ret transgenic mice, we showed a strong tumor infiltration with immature DC, which secreted significantly more interleukin 10 and less interleukin 12p70, and showed a decreased capacity to activate T cells (7). Observed DC dysfunction was linked to considerable activation of p38MAPK. The aim of the project is to investigate the anti-tumor effects of selective inhibitors of MAPK in DC as well as the possibility to restore T-cell activities in vitro and in vivo. In addition, appropriately activated DC loaded with melanoma antigen are planned to be adoptively transferred into melanoma bearing mice alone or in combination with re-stimulated MAA-reactive memory T cells (see also project 1).
Within this topic we have a small project to be done within a medical thesis or master thesis. If you are interested in this project, please use the contact form of Viktor Umansky.

Project 6: Development of combined chemotherapy and immunotherapy of melanoma using ret transgenic mouse melanoma model

Standard melanoma chemotherapy is not satisfactory and may increase tumor-mediated immunosuppression. In contrast, low dose chemotherapy can induce immunogenic tumor cell death and stimulate antitumor immune responses by efferocytosis. Efferocytosis is immunogenic way of apoptosis which characterizes with early calreticulin (CRT) expression before phosphatidylserine expression on cell surface. The goal of the project is to study the molecular mechanism of efferocytosis induction by cyclophosphamide and temozolomide in low doses and of their effects on immune cells (in particular, effector T cells, Tregs and myeloid derived suppressor cells). Application of this chemotherapy could increase the immunogenicity of tumor cells, inhibit immunosuppressive cell activity and induce therefore an antitumor effect.

Cooperations

• Prof. Dr. S. Eichmüller, Dr. W. Osen (same department)
• PD Dr. Christine Falk (DKFZ, G101)
• Prof. Dr. P. Beckhove (DKFZ, D015)
• Dr. N. Garbi (DKFZ, D040)
• Dr. A. Bazhin (University Heidelberg)
• PD Dr. K. Mahnke (University Heidelberg)
• Prof. J. Becker (University Würzburg)
• Prof. H. Schild (University Mainz)
• Dr. M. Baniyash (Jerusalem, Israel)
• Dr. T. Schumacher (Amsterdam, Netherlands)
• Dr. C. Leclerc (Paris, France)
• Dr. M. Shurin (Pittsburgh, USA)
• Dr. I. Borrello (Baltimore, USA)
• Dr. R. Offringa (Leiden, Netherlands)
• Dr. M. Kato (Aichi, Japan)