Phd in IINNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Thesis title: Granzyme A and CD160 expression delineates ILC1 with graded functions in the mouse liver

Type 1 innate lymphoid cells (ILC1) are tissue-resident lymphocytes which provide early protection against bacterial and viral infections. Discrete transcriptional states of ILC1 have been identified within the same organ in homeostatic and pathological contexts. However, whether these states are associated with different functional properties needs to be evaluated. Thus, the aim of my Ph.D research work was to deconvolute ILC1 heterogeneity and to assess a functional diversification of distinct liver ILC1 subsets. First, we demonstrated that liver ILC1 displayed a heterogeneous expression of distinct effector molecules and surface receptors, such as granzyme A (GzmA) and CD160, by performing single cell RNA-seq analysis and confirming transcriptomic data through flow cytometry approaches. In particular, GzmA+ ILC1 were found mainly in the liver rather than other peripheral organs. Moreover, we observed that CD160+GzmA+ ILC1 represented the main hepatic ILC1 population at birth and the expression pattern of CD160 and GzmA was gradually established throughout adulthood. Secondly, we found that GzmA+ ILC1 differed from NK cells for the requirements of JAK/STAT signals and the transcription factor Nfil3. In addition, experiments of adoptive transfer and in vitro stimulation showed that NK cells had limited capacity to give rise to GzmA+ ILC1 both in homeostatic condition and upon TGF-b exposure. On the other hand, by employing Rorc(gt)-fate map (fm) reporter mice, we established that ILC3-ILC1 plasticity contributed to delineate the heterogeneity of liver ILC1, with Rorgt-fm+ cells skewed towards a GzmA-CD160+ phenotype. Finally, we showed that ILC1 defined by the expression of GzmA and CD160 were characterized by a graded cytotoxic potential and ability to produce IFN-g, by employing a flow cytometry-based killing assay with YAC-1 cells and cytokines stimulation, respectively. Collectively, our data provide evidence for a spectrum of phenotypically and functionally distinct ILC1 subsets. Understanding the modality for liver ILC1 to contribute to the immune response can be of relevance in the context of liver pathology.