Identification of a Myeloid Pathway of NK Cell Development and a Myeloid- NK Cell Precursor
Authors: Grzywacz Bartosz , Kataria Nandini , Blazar Bruce R. , Miller Jeffrey S. , Verneris Michael 
Published: April 2020

Abstract
NK cells are CD56+CD3− innate immune effector cells that play important roles in tumor surveillance. Since the identification of a common lymphoid progenitor (CLP) and the demonstration that NK cells can be derived from CLPs, the ontogeny of NK cells has been assumed to be exclusively lymphoid. Our laboratory has been studying NK cell development using CD34+ hematopoietic progenitors (HPCs) cultured on a murine fetal liver stromal cell line (EL08.1D2) in the presence of cytokines (IL-3 for the first week, IL-7, IL-15, SCF and FLT-3L) and hydrocortisone (HDC). After 3–4 weeks under these conditions, NK cells develop. We set out to determine the impact of stroma and HDC on NK cell development. Previous studies had identified antigens on CD34 HPCs that were associated with a greater propensity to become NK cells when cultured in cytokines (including CD7, CD161, CD45RA and intergrin b7). FACS purifying CD34 cells that lacked these receptors (CD34+NKlin−) identified a subset of CD34 cells that was absolutely dependent on stroma or HDC for functional NK cell development (using limiting dilution assays, p&lt;0.05). In the absence of stroma and HDC, however, CD34+NKlin− cells showed myeloid lineage (CD13 and CD33) outgrowth. Likewise, under the influence of cytokines, HDC and stroma, CD34+NKlin− cells appeared to first acquire CD13 and/or CD33, then CD56. These results lead us to hypothesize that the mechanism of HDC and stroma was to “skew” myeloid cells to the NK lineage. To test this, CD34+NKlin− cells were cultured for 2 wks and CD13low/−, CD13int and CD13high cells were FASC sorted. Culture of sorted populations with cytokines alone did not give rise to NK cells. The addition of either HDC or stroma resulted in the development of NK cells and their combination was additive (p&lt;0.05). Importantly, there was a quantitative difference in NK cell generation from the three fractions, with decreasing NK cell growth with advancing myeloid commitment. In further experiments CD34+NKlin− HPCs were cultured for two weeks and then CD14+ cells were isolated. NK cells again developed with the combination of cytokines, HDC and stroma, but not with cytokines alone. To further query whether myeloid cells could become NK cells, we isolated CFU-GMs from 3 wk methylcellulose colonies and cultured them with cytokines or cytokines, HDC and stroma. NK cell developed only in the presence of cytokines, HDC and stroma (0/36 vs. 9/51, p&lt;0.05). Collectively, these results suggested a myeloid-NK progenitor. The M-CSF receptor (M-CSFR, CD115) is expressed by granulo-monocytic precursors, promoting monocytic differentiation in response to M-CSF. Therefore, M-CSFR is not merely a phenotypic marker, but is functionally linked with the monocytic differentiation. CD117highCD56−CD115+ and CD115− cells were isolated and tested for their ability to give rise to NK cells. Strikingly, both could give rise to NK cells, but CD115+ progenitors required HDC and stroma for full NK cell maturation. Addition of high doses of M-CSF to CD115+ progenitors abolished NK cell development. Lower doses of M-CSF lead to NK cells that co-expressed CD14, supporting the existence of a common NK-myeloid progenitor. Lastly, the NK cells derived from the myeloid progenitors (CD117highCD56−CD115+) were compared to the remaining “lymphoid” fraction. The myeloid derived NK cells expressed significantly more KIR receptors and had remarkably higher killing against B-LCL cells (721-221) (p&lt;0.05 for both, n=4). Collectively, these studies show that cells at seemingly late stages of myeloid development cells can become NK cells under the influence of stroma and HDC. Thus, like dendritic cells, NK cells can be derived from both myeloid and lymphoid developmental pathways. Such NK cells differ both functionally and phenotypically depending upon their developmental origin. Lastly, we demonstrate the existence of a previously unknown common myeloid-NK progenitor (CD117highCD56−CD115+).