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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.)

Innate lymphoid cells (ILCs) and in particular ILC3s have been described to be vital for mucosal barrier functions and homeostasis within the gastrointestinal (GI) tract. Importantly, IL-22-secreting ILC3 have been implicated in the control of inflammatory bowel disease (IBD) and were shown to reduce the incidence of graft-versus-host disease (GvHD) as well as the risk of transplant rejection. Unfortunately, IL-22-secreting ILC3 are primarily located in mucosal tissues and are not found within the circulation, making access to them in humans challenging. On this account, there is a growing desire for clinically applicable protocols for in vitro generation of effector ILC3. Here, we present an approach for faithful generation of functionally competent human ILC3s from cord blood-derived CD34 + hematopoietic progenitors on layers of human mesenchymal stem cells (MSCs) generated in good manufacturing practice (GMP) quality. The in vitro -generated ILC3s phenotypically, functionally, and transcriptionally resemble bona fide tissue ILC3 with high expression of the transcription factors (TF) RorγT, AHR, and ID2, as well as the surface receptors CD117, CD56, and NKp44. Importantly, the majority of ILC3 belonged to the desired effector subtype with high IL-22 and low IL-17 production. The protocol thus combines the advantages of avoiding xenogeneic components, which were necessary in previous protocols, with a high propensity for generation of IL-22-producing ILC3. The present approach is suitable for the generation of large amounts of ILC3 in an all-human system, which could facilitate development of clinical strategies for ILC3-based therapy in inflammatory diseases and cancer., Competing Interests: The 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 © 2021 Bennstein, Weinhold, Degistirici, Oostendorp, Raba, Kögler, Meisel, Walter and Uhrberg.)

Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Natural killer T-cells are a subset of innate-like T-cells with the ability to bridge innate and adaptive immunity. There is great interest in harnessing these cells to improve tumor therapy; however, greater understanding of invariant NKT (iNKT) cell biology is needed. The first step is to learn more about NKT development within the thymus. Recent studies suggest lineage separation of murine iNKT cells into iNKT1, iNKT2, and iNKT17 cells instead of shared developmental stages. This review will focus on these new studies and will discuss the evidence for lineage separation in contrast to shared developmental stages. The author will also highlight the classifications of murine iNKT cells according to identified transcription factors and cytokine production, and will discuss transcriptional and posttranscriptional regulations, and the role of mammalian target of rapamycin. Finally, the importance of these findings for human cancer therapy will be briefly discussed.