Human IL-22 Producing CD56⁺ RORγt⁺ Innate Lymphoid Cells and Conventional NK Cells Have Distinctive Developmental Trajectories and Are Separate Cell Lineages

Abstract 1219

Human IL-22 producing RORγt⁺ innate lymphoid cells (ILC22) and conventional NK (cNK) cells are present in secondary lymphoid tissues. Both cell types have an immunophenotype that correspond to stage III NK progenitors (CD56^+/−CD117^highCD94⁻), leading us and others to speculate that the IL-22 producing cells are part of the NK lineage and can give rise to cNK cells (Tang, Blood, 2010, Cupedo, Nat Imm, 2009 and Colona, Immunity, 2009). However, recent fate mapping studies in mice suggest that these cell types are separate lineages (Sawa, Science 2010). Given the significant phenotypic and functional differences between human and murine ILC22 cells, this issue is unresolved in humans. To address this, we used an established differentiation system where UCB-derived CD34⁺ cells are cultured on irradiated fetal liver stromal cells in the presence of IL-3 (5 ng/ml, for the first week), IL-7 (20 ng/ml), SCF (20 ng/ml), FLT3L (10 ng/ml) and IL-15 (10 ng/ml). We have previously demonstrated that this model precisely recapitulates NK cell developmental intermediates, as well as IL-22 producing ILCs (Gryzwacz, Blood, 2005 and Tang, Blood, 2011). We first set out to determine whether it was possible to distinguish IL-22 producing ILCs from cNK using intracellular cytokine staining and a panel of mAbs. Non-IL-22 producing cNK cells showed a CD56⁺CD117^lo/-CD7^+/−LFA-1^high phenotype, while ILC22 cells were completely contained within the CD56⁺CD117^highCD94⁻CD7⁻LFA-1⁻ fraction. Purification of these two populations showed that ILC22 cells expressed high quantities of transcription factors associated with IL-22 production including AhR and RORγt, while these were absent or barely detectable in cNK cells (p<0.0001). Conversely, T-bet and Eomes were highly expressed in cNK progenitors, but not ILC22 cells. While cNK cells expressed granzyme and perforin, classical NK-associated receptors (NKp30, NKp46, NKG2A, NKG2D, CD8, CD16 and KIR) and showed degranulation (CD107a) and produced IFN-γ in response to K562 targets or IL-12+IL-18, ILC22 cells did not. Thus, ILC22 and cNK cells were distinguishable by transcription factor expression, surface receptor expression and function. To investigate the lineage relationship between ILC22 cells and cNK cells, stage III NK progenitors (defined as CD56⁺CD117⁺CD94⁻) were purified on the basis of LFA-1 expression and then further cultured. Cells that expressed LFA-1 (i.e., cNK progenitor cells) rapidly acquired CD94, and differentiated into stage IV and V cNK cells. Conversely, the vast majority of cells that lacked LFA-1 cells (i.e., ILC22 cells) acquired neither LFA-1 nor CD94, thus never differentiate into stage IV and V cNK cells. These results suggest that ILC22 cells represent a separated and stable cell lineage from cNK cells. To further address this and investigate the developmental requirements for cNK and ILC22 cells, CD34⁺ hematopoietic stem cells were cultured in the above conditions with or without IL-7 and SCF, which are known to be critical cytokines for lymphoid tissue inducer (LTi) cell generation in vivo (a population similar to ILC22 cells). In the absence of IL-7 and SCF, cNK cells developed normally while ILC22 cells did not develop. These results show that cNK cells differentiated even in the absence of ILC22 stage III cells, which require SCF and IL-7 for differentiation. Conversely, in the absence of IL-15, CD34⁺ cells showed a complete block in cNK differentiation and instead gave rise to a CD56⁺ILC22 cells, and their phenotype and function were normal. Thus, while human ILC22 cells and cNK progenitors have a phenotype that overlaps with stage III NK progenitors, these studies demonstrate that they are separate cell lineages, with differing phenotype, transcription factor expression, developmental requirements and functions.