Umbilical cord blood-derived ILC1-like cells constitute a novel precursor for mature KIR+NKG2A- NK cells
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Abstract
Despite their identification several years ago, molecular identity and developmental relation between human ILC1 and NK cells, comprising group 1 ILCs, is still elusive. To unravel their connection, thorough transcriptional, epigenetic, and functional characterization was performed from umbilical cord blood (CB). Unexpectedly, ILC1-like cells lacked Tbet expression and failed to produce IFNγ. Moreover, in contrast to previously described ILC1 subsets they could be efficiently differentiated into NK cells. These were characterized by highly diversified KIR repertoires including late stage NKG2A-KIR+ effector cells that are commonly not generated from previously known NK cell progenitor sources. This property was dependent on stroma cell-derived Notch ligands. The frequency of the novel ILC1-like NK cell progenitor (NKP) significantly declined in CB from early to late gestational age. The study supports a model in which circulating fetal ILC1-like NKPs travel to secondary lymphoid tissues to initiate the formation of diversified NK cell repertoires after birth.
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###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on March 4, 2020, follows.
Summary
In this study, Bennstein et al describe a T-bet negative ILC1-like cell population identified in cord blood. These cells lack the signature of NK cell genes and markers, and instead express genes and markers of T-cell lineage. In appropriate culture conditions these cells can differentiate into a complex repertoire of functional NK cells that express both NKG2A and diverse KIRs. The reviewers appreciated the attention to an important topic, but raised a substantial number of concerns about the manuscript as it currently stands. We therefore ask the authors to modify the manuscript according to the review recommendations.
Essential Revisions
The authors state that ILC1-like cells which are T-bet and …
###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on March 4, 2020, follows.
Summary
In this study, Bennstein et al describe a T-bet negative ILC1-like cell population identified in cord blood. These cells lack the signature of NK cell genes and markers, and instead express genes and markers of T-cell lineage. In appropriate culture conditions these cells can differentiate into a complex repertoire of functional NK cells that express both NKG2A and diverse KIRs. The reviewers appreciated the attention to an important topic, but raised a substantial number of concerns about the manuscript as it currently stands. We therefore ask the authors to modify the manuscript according to the review recommendations.
Essential Revisions
The authors state that ILC1-like cells which are T-bet and CD56 negative and lack expression of perforin and all 5 granzymes develop into effector NK cells with de novo CD94, NKG2A and KIR2DL3 expression. The authors need to further phenotype the differentiated cells, showing evidence of essential NK cell markers, including CD56, NKp46, granzyme B and perforin. Additionally, they should demonstrate that inhibitory KIR expression in the differentiated cells is functionally inhibitory and leads to increased cytotoxicity in educated cells. Despite the ILC1-like derived NK cells having increased KIR expression compared to the CD56bright-derived NK cells, CD56bright-derived NK cells were equally functional and exhibited more target-specific degranulation compared to ILC1-derived NK cells.
The idea proposed in the discussion that CB ILC1 may be cells that have failed to convert into T cells into the thymus is an attractive one and perhaps the authors may wish to test it by looking at markers of recent thymic emigrants in CB ILC1 - if possible?
It is unclear if proper functional controls were utilized in this study. Target-specific degranulation needs to be shown instead of total degranulation in fig 5, as fig 7c makes evident. Additionally, the equation used to calculate cytotoxicity for the CFSE-based method should be included in the materials & methods section. For the ADCC assay, it is unclear what cells were used for the control. Individual controls (antibody negative) for each cell population should be included.
How do the authors explain the phenotypic and functional differences between the individual ILC1-like subsets as defined by CD5 and CD161 expression and how do these individual subsets contribute to their proposed NKP potential? This point should be discussed in more detail.
The authors state that ILC1-like cells preferentially differentiate into mature KIR+ NK cells compared to CD56bright NK cells in the OP9-DL1 differentiation setting in the presence of IL-2, IL7 and IL-15. Cytokine stimulation (IL-2 and IL-15) of NK cells leading to the induction of proliferation and results in CD56 and NGK2A upregulation, even in mature CD56dim NK cells. Hence it is not surprising that CD56bright NK cells retained high NKG2A expression while actively proliferating. The present experimental setup therefore does not support their statement on line 570-573 of a branched NK cell lineage model.
The authors clearly show that the CD127+ Lin- population is highly heterogenous, just by looking at CD161, CD5, CCR9, CCR4 and CCR7. Therefore, the transcriptomic and epigenetic data on the bulk population are not informative. Single cell analysis should be used to define the heterogeneity, considering that Simoni et al. have previously reported the heterogenous nature of human ILC1s (Simoni et al., Immunity, 2017) and questioned the nature of lineages included.
The authors claim that the cells do not generate T cells. However, they only use IL-7 and FLT3, while in other protocols the used IL-7, FLT3L and SCF.
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