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.

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