Cellular Immunology

High-resolution barcoding

To decipher hematopoiesis and the immune system at single cell and clonal levels our laboratory has developed Cre-loxP-based DNA and RNA barcoding technologies, termed 'Polylox' and 'PolyloxExpress', respectively (Pei, Feyerabend et al. Nature 2017; Pei, Wang, Rössler et al. Nature Protocols 2019; Pei, Shang, Wang et al. Cell Stem Cell 2020; Fanti, Busch et al. Cell Stem Cell 2023). 

Application of these new endogenous barcoding technologies led, among other findings, to the classification of HSC fates into distinct classes, i.e. multilineage, myeloid-erythroid restricted, and differentiation-inactive. The second generation of our barcoding system, 'PolyloxExpress' facilitates simultaneous recovery of barcodes and transcriptomic information at the single cell level. This allowed us to directly link cell fate output with underlying transcriptomic signatures (Pei, Shang, Wang et al. Cell Stem Cell 2020). 

We designed ‘Polytope’ to link spatial information and clonal lineage tracing in situ (Postrach et al. bioRxiv 2024). Polytope has 512 distinct color codes, which can be visualized by conventional fluorescence microscopy, providing a comprehensive tool for capturing complex clonal dynamics in situ. For proof of principle, we traced the fate of hundreds of clones across tissues from embryonic development to adulthood. 

Overall, our ongoing and future work aims to apply these technologies to further dissect general mechanisms of hematopoietic progenitor cell fate decisions in a multi-omics approach and specifically developmental ontogenies of tissue-resident immune cell subsets, such as macrophages (Frank, Postrach, Langhinrichs et al. bioRxiv 2024), and innate lymphoid cells (Cirovic et al. in preparation) in situ.

Leukemia development and progression

In previous work, we identified cell competition between 'young' bone marrow-derived and 'old' thymus-resident progenitors as a tumor suppressor mechanism for T-cell acute lymphoblastic leukemia (T-ALL) (Martins et al. Nature 2014). Disruption of new influx of progenitors into the thymus leads to spontaneous development of full-blown T-ALL and provides a unique and robust system to unravel cellular and molecular events along the transition from normal to leukemic T cells, closely resembling the human disease (Martins et al. Nature 2014; Ginn et al. Mol Ther Nucleic Acids 2017; Schiroli et al. Sci Transl Med 2017; Gao J et al. Dis Model Mech 2019). As this tumor model is independent of a priori introduced oncogenes, it makes the 'natural emergence' of clones and mutations kinetically accessible and, in general, presents arguably a unique tool to address mechanisms of tumor development and progression.

Along these lines, we apply this system of tumor development in the following contexts:

• Investigation of Notch1 driver mutations as rate-limiting step of tumorigenesis

• Modeling of stem cell output on the risk of acquiring oncogenic drivers and leukemia progression (in collaboration with the laboratory of Thomas Höfer)

• Deconvolution of metastasis behavior of T-ALL by applying Polylox and PolyloxExpress barcoding and lineage tracing