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The generation and expansion of functionally competent NK cells in vitro is of great interest for their application in immunotherapy of cancer. Since CD33 constitutes a promising target for immunotherapy of myeloid malignancies, NK cells expressing a CD33-specific chimeric antigen receptor (CAR) were generated. Unexpectedly, we noted that CD33-CAR NK cells could not be efficiently expanded in vitro due to a fratricide-like process in which CD33-CAR NK cells killed other CD33-CAR NK cells that had upregulated CD33 in culture. This upregulation was dependent on the stimulation protocol and encompassed up to 50% of NK cells including CD56 dim NK cells that do generally not express CD33 in vivo . RNAseq analysis revealed that upregulation of CD33 + NK cells was accompanied by a unique transcriptional signature combining features of canonical CD56 bright (CD117 high , CD16 low ) and CD56 dim NK cells (high expression of granzyme B and perforin). CD33 + NK cells exhibited significantly higher mobilization of cytotoxic granula and comparable levels of cytotoxicity against different leukemic target cells compared to the CD33 - subset. Moreover, CD33 + NK cells showed superior production of IFNγ and TNFα, whereas CD33 - NK cells exerted increased antibody-dependent cellular cytotoxicity (ADCC). In summary, the study delineates a novel functional divergence between NK cell subsets upon in vitro stimulation that is marked by CD33 expression. By choosing suitable stimulation protocols, it is possible to preferentially generate CD33 + NK cells combining efficient target cell killing and cytokine production, or alternatively CD33 - NK cells, which produce less cytokines but are more efficient in antibody-dependent applications., Competing Interests: CZ, MQ, MN, SC, RP, and NM are employees of Miltenyi Biotec. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hejazi, Zhang, Bennstein, Balz, Reusing, Quadflieg, Hoerster, Heinrichs, Hanenberg, Oberbeck, Nitsche, Cramer, Pfeifer, Oberoi, Rühl, Oldenburg, Brossart, Horn, Babor, Wels, Fischer, Möker and Uhrberg.)

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., Competing Interests: SB, SW, AM, NS, AN, KR, GK, LW, MU No competing interests declared, (© 2020, Bennstein et al.)