CONTROL-T RESEARCH PROJECT 2 (active from 2013-2016)
First Funding Period
The team (as of August 2013)
From left:Prof. Martin-Leo Hansmann
Dr. Benjamin Rengstl
Dr. Kathrin Warner
Dr. Frederike Schmid
Christian Weiser
Dr. Sebastian Newrzela
Abstract
To assess the capacity of mature T cells to become oncogenetically transformed we overexpressed different T-cell (proto-)oncogenes in mature T cells or hematopoietic stem cells/progenitor cells (HSC/HPC). We found that transduced HSC/HPC readily induced T-cell leukemia/lymphoma in mice. However, mature T-cell receptor (TCR) polyclonal T cells could not be transformed. Quite surprisingly, TCR quasi-monoclonal T lymphocytes (OT-I) were transformed with the same kinetics and efficiency as stem cells. Within an ongoing DFG project (LA1135/9-1) we could transform a further TCR monoclonal T-cell type (P14), indicating that this observation is a more general phenomenon. Moreover, we found that addition of polyclonal T-cell populations prevented malignancies and we excluded immunological processes as a controlling factor. As it is described for normal T-cell homeostasis, we hypothesize, that outgrowth of pre-leukemic clones is controlled by the competition of different T-cell clones for stimulatory MHC-self-peptide niches, an underlying hypothesis of several projects within the proposed research unit (CONTROL-T). Our experimental observations clearly demonstrate that outgrowth of peripheral T-cell tumors is prevented by TCR-polyclonality. We plan to analyze the population-dynamics in leukemia/lymphoma development and to elucidate the molecular mechanisms that control the development of T-cell malignancies in the TCR-polyclonal scenario. Furthermore, our experimental system is highly relevant for evaluating the safety of retroviral vector-based T-cell gene therapy.Aims
Objectives:
Investigating the potential role of homeostatic clonal competition in the control of lymphomagenesis is a demanding task. The proposed research project aims to understand clonal dynamics of oncogene-modified TCR polyclonal and TCR monoclonal T-cell populations. We want to shed light on the molecular mechanisms that control clone size in normal T-cell homeostasis and that are lost during pathogenesis of MTCL.The following specific objectives will be tackled:
Aim 1
We will investigate the overall dynamics of WT (TCR polyclonal) and TCR transgenic (tg) (TCR monoclonal) T-cell populations after transplantation into lymphopenic RAG-1 deficient recipients. Hereby, oncogene or control-gene transduced murine T cells of WT and tg donors will be studied ex vivo. We want to determine important key features of the different T-cell populations in our experimental setting.Aim 2
We aim to identify and validate pivotal molecular pathways/players that control malignant clonal outgrowth in TCR polyclonal T-cell populations.Aim 3
To elucidate clonal dynamics during lymphomagenesis, we want to retrospectively analyze the phenotype and the fluctuation/frequency of outgrowing malignant clones during tumor development.Preliminary Data
Over the past years, the group has developed murine T-cell and stem cell transplantation models that involve ex vivo genetic modification with retroviral vectors. We could show that mature TCR polyclonal T cells are relatively resistant to transformation, while TCR quasi-monoclonal T cells can be transformed by overexpression of specific T-cell oncogenes.TCR polyclonal mature T cells demonstrate a relative resistance to oncogene transformation
Previously, we analyzed whether RV-based gene transfer into mature T cells bears the same genotoxic risk as described for HSCs (Newrzela et al., 2008). In a comparative study, we generated an experimental “worst-case-scenario” by transducing mature, TCR polyclonal T cells and HSCs from C57BL/6 (Ly5.1) donor mice with high copy numbers of RVs encoding the potent T-cell oncogenes LMO2, TCL1, or ΔTrkA, a constitutively active mutant of TrkA. After transplantation into RAG-1-deficient recipients (Ly5.2), stem cell transplanted animals developed T-cell lymphoma/leukemia for all investigated oncogenes with a characteristic phenotype and after characteristic latencies (see Figure 1). Ligation-mediated PCR analysis revealed mono- or oligoclonality of the malignancies. In striking contrast, none of the mice transplanted with mature T cells transduced with the same vectors developed leukemia/lymphoma despite long-term persistence of gene-modified cells that were expressing high levels of the oncogene. Thus, the data of this study clearly showed that mature, TCR polyclonal T cells are less prone to transformation than HSCs. Additionally, in two further studies based on RV-mediated modification of TCR polyclonal T cells, we never observed any malignant clonal outgrowth after transplantation (Preuss et al., 2010; Vogler et al., 2010).
Figure 1: Mature TCR polyclonal T cells are less susceptible to transformation by T-cell oncogenes than HSCs. Animals transplanted with HSCs transduced with the oncogenes LMO2 (A), TCL1 (B), ΔTrkAhigh (C) or ΔTrkAlow (D) developed hematological malignancies after characteristic latencies (dashed lines, A-D). Control animals transplanted with MP91-EGFP (E) and recipients of TCR-polyclonal T-cell transplants survived throughout the whole observation period (continuous lines, A-E) with no sign of leukemia/lymphoma.
Retroviral insertional mutagenesis can contribute to immortalization of mature T Lymphocytes
Simultaneously, in an in vitro study, we observed immortalization of a single T-cell clone after retroviral transduction of the T-cell proto-oncogene LMO2 (Newrzela et al., 2011). This clone was CD4/CD8 double-negative, but expressed a single rearranged TCR. The clone was able to overgrow non-manipulated competitor T-cell populations in vitro (Figure 2), but no tumor formation was observed after transplantation into RAG-1 deficient recipients. The retroviral integration site (RIS) was found to be near the IL2RA and IL15RA genes. As a consequence, both receptor chains were constitutively upregulated on the RNA and protein level and the immortalized cell clone was highly IL-2 dependent. Ectopic expression of both, the IL2RA chain and LMO2, induced long-term growth in cultured primary T cells. In this study we demonstrated that insertional mutagenesis can contribute to immortalization of mature T cells, although this is a rare event. Furthermore, the results indicated that signaling of the IL-2 receptor and the proto-oncogene LMO2 can act synergistically in malignant transformation of mature T lymphocytes.
Figure 2: Competition experiment with immortalized mature T cells. Freshly stimulated T cells were chosen as competitors and set to 90% in the mixed culture, the remaining 10% were immortalized, EGFP-expressing cells (d 0). Flow cytometry analysis was performed on the days: 2, 6 and 8. The immortalized T cells eventually overgrew the competitor cells on day 8 of culture.
A background of TCR polyclonality prevents MTCL development
Based on the observations described above, we hypothesize that clonal competition for stimulatory pMHC niches among mature TCR polyclonal T cells potentially controls lymphomagenesis. To corroborate this idea, we introduced the oncogenes NPM/ALK or ΔTrkA into TCR quasi-monoclonal mature T cells from TCR transgenic (tg) mice (OT-I, P14). Transplanted RAG-1-deficient recipients readily developed MTCLs (Newrzela et al., 2012). Analysis of cell surface markers largely ruled out that TCR tg lymphomas were derived from T-cell precursors. Furthermore, co-transplanted non-modified wild-type (WT) TCR polyclonal T cells suppressed malignant outgrowth of oncogene expressing TCR tg T lymphocytes (Figure 3A). A dominant role of an anti-leukemic immune response or regulatory T cells in the control of MTCLs was ruled out, as naïve TCR polyclonal T cells derived from oncogene expressing stem cells, which should be tolerant to leukemic antigens, as well as purified TCR polyclonal CD4+ or CD8+ T cells were resistant to transformation. However, our results are in line with a model, in which homeostatic mechanisms that normally stabilize the diversity of the T-cell repertoire, e.g. clonal competition, may have suppressed the outgrowth of potentially malignant T-cell clones.
Figure 3: TCR polyclonality suppresses MTCL. (A) Recipients of NPM/ALK TCR tg OT-I T cells developed MTCL. TCR polyclonal WT T cells developed mature T-cell lymphoma with a delayed onset and animals transplanted with TCR polyclonal WT competitor T cells survived without any indication of lymphoma. (B) TCR did not induce any malignancy.