CD33 Delineates Two Functionally Distinct NK Cell Populations Divergent in Cytokine Production and Antibody-Mediated Cellular Cytotoxicity.

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.)