Entschlüsselung von Gedächtnis-artigen NK-Zellantworten mit Hilfe von Polylox-Barcoding
Zusammenfassung der Projektergebnisse
In this project, we attempted to further our understanding of memory-like responses mounted by NK cells during MCMV infection. True immunological memory must fulfill certain criteria: 1) The expansion must be antigen specific. 2) The memory response must be clonal, which means that the same cell must mount acute expansion and the memory response. While the first requirement has been established for NK cells in MCMV infection, the question whether NK cell memory is clonal remains mostly unanswered. To resolve NK cell expansion in clonal resolution, we have used retrogenic color-barcoding in the past8,16. However, retrogenic color-barcoding has certain limitations. For example, so far it has only worked in severely immunocompromised hosts. Endogenous barcoding using the polylox approach is capable of a high-throughput analysis of clonal composition in immunocompetent mice. Therefore, we wanted to establish this method for the study of NK cell memory-like responses. We succeeded in performing a first experiment with polylox-barcoded NK cells. We could find that the same NK cell clones can contribute both to the acute and memory-like responses. It appeared that not all clones found during the acute expansion contributed to the pool of memory-like NK cells. One possibility that could explain why certain NK cell clones mount memory-like responses and others do not could be that clones differ in their ability to home into different organs. The liver has been implicated as crucial for NK memory-like responses in other settings11. When analyzing the clonal composition in different organs following MCMV infection, we could however not find a striking difference in the abundance of certain NK cell clones in different organs. Rather, it appeared like clones that were most prevalent in the spleen were also found in higher abundance in liver and lungs. Overall, the number of clones that we could analyze in our first experiments was too low to draw final conclusions. Technically, one challenge was that polylox-barcodes were over-recombined, yielding a low number of high-quality barcodes. We could optimize this by titrating down the dose of Tamoxifen, which is required for translocation of Cre into the nucleus in NK-CreERT2 x polylox mice. Titrating the tamoxifen dose too as low as 0.1mg/mouse yielded optimal barcode composition. With this optimized protocol we will attempt to increase the number of NK cell clones analyzed in currently ongoing experiments. We complemented our clonal study of adaptive NK cell responses with further experiments that sought to address 1) whether memory-like NK cell responses are mounted primarily by certain subsets of NK cells found in naïve mice and 2) whether NK cells require the transcription factor TCF7 for memory-like responses – a transcription factor that has been shown to be crucial for CD8+ T cell memory. Regarding the first question, we could find that the most immature NK cells during steady state are best both at mounting both acute and memory responses. This does not exclude the possibility that within this compartment different NK cell phenotypes differ in their capacity to mount memory-like responses specifically. Most crucially, in a recent study by us we identify ILC1-like NK cells within this most immature subset to be a stable phenotype. In future experiments, we will transfer immature and ILC1-like NK cells separately for the clonal study of NK cell memory. Next, we addressed whether TCF7 is required for longevity of NK cells in adaptive responses similarly to CD8+ T cells. If this were the case, a differential expression of this transcription factor in different subsets could help us identify NK memory precursor subsets which was the main aim for this project. In co-transfer experiments, we found that TCF7 was required for optimal NK cell expansion, but memory-like responses did not fundamentally differ between wildtype and TCF7-deficient NK cells. Together, our data suggest that polylox barcoding can be a valuable tool for the study of memorylike responses mounted by NK cells in MCMV infection. Further experiments that will help identify whether there is a specific memory-precursor subset are currently underway. We hope to be able to submit this study for publication within the next 1-2 years.
Projektbezogene Publikationen (Auswahl)
- Clonal expansion of innate and adaptive lymphocytes. Nature Reviews Immunology 20, 694-707
Adams, N. M., Grassmann, S. & Sun, J. C.
(Siehe online unter https://doi.org/10.1038/s41577-020-0307-4) - Divergent Role for STAT5 in the Adaptive Responses of Natural Killer Cells. Cell Reports, Volume 33, Issue 11 (2020)
Wiedemann, G.M., Grassmann, S., Lau C.M. et al.
(Siehe online unter https://doi.org/10.1016/j.celrep.2020.108498) - Early emergence of T central memory precursors programs clonal dominance during chronic viral infection. Nature Immunology volume 21, pages 1563–1573 (2020)
Grassmann, S., Mihatsch, L. et al.
(Siehe online unter https://doi.org/10.1038/s41590-020-00807-y) - Deconvoluting global cytokine signaling networks in natural killer cells. Nature Immunol 22, 627–638 (2021)
Wiedemann, G.M., Santosa, E.K., Grassmann, S. et al.
(Siehe online unter https://doi.org/10.1038/s41590-021-00909-1) - Fate mapping of single NK cells identifies a type 1 innate lymphoid-like lineage that bridges innate and adaptive recognition of viral infection. Immunity, Oct 12;54(10):2288-2304.e7
Flommersfeld, S., Boettcher J., (…) Buchholz, V.R. and Grassmann, S.
(Siehe online unter https://doi.org/10.1016/j.immuni.2021.08.002)
