(J-376) CD2 Regulates Cellular Cytotoxicity Mediated By Natural Killer Cells

Many advances have been made recently in developing induced pluripotent stem cell (iPSC)-derived NK cell therapies. However, many of these iPSC NK cells do not express CD2, which may limit NK cytotoxicity. NK cells are cytotoxic lymphocytes that are part of the innate immune system responsible for killing virally infected and cancer cells. NK cells become activated when the total activation signal exceeds the total inhibitory signal [1]. Once activation is reached, NK cells create an immunological synapse (IS) with the target cell and secrete lytic granules containing perforin and granzymes directly toward the target cell, resulting in cell death [2]. CD2 is a potential co-receptor for NK cells. In T cells, CD2 has been shown to polarize toward the cell-cell contact site, where it helps with the assembly and organization of the IS and positively regulates TCR signaling intensity. In NK cells, CD2 has been found to play a role in NK cell conjugation, recruitment of CD16 to the IS, and interaction with CD16 to enhance antibody-mediated cellular cytotoxicity. Although the function of CD2 in NK cells and T cells seems similar, there is very little research on the function of CD2 in NK cells [3]. This research aims to further understand the role of CD2 in NK cell cytotoxicity in order to determine the importance for iPSC NK cells to express this receptor. 

For rADCC assays, primary NK cells were incubated for 3 hr with 51Cr-labeled target cells at various Effector: Target (E: T) ratios, and the percent specific lysis was calculated. Target cells were P815 coated with 0.5 ug anti-CD2 and/or 0.25 ug anti-CD16. pNK CD2 knock-out (KO) cells were created by performing two knock-outs of the CD2 gene on primary NK cells. Flow cytometry was performed to determine the percentage knock-out of CD2. The CD2 KO cells were used in killing assays where pNK NC and KO cells were incubated for 3 hr with 51Cr-labeled target cells at various E: T ratios, and the percent specific lysis was calculated. The target cells were Raji cells and four pancreatic ductal adenocarcinoma (PDAC) cell lines: L3.6, 03.27, Panc1, and 4535. The CD2 KO cells were also used in ADCC assays where pNK NC and KO cells were coated with daratumumab (an anti-CD38 antibody) at 10ug/mL, incubated for 3 hr with 51Cr-labeled Raji cells at various E:T ratios, and the percent (%) specific lysis was calculated. Stimulation assays were performed by stimulating pNK cells with anti-CD2, anti-CD16, or both for 0, 2.5, 5, and 10 minutes. Western blots were then stained for levels of phosphorylated Erk (pERK), total Erk, and phosphorylated tyrosine. The coverslips were created by co-incubating PDAC and primary NK cells on coverslips for 20 minutes, fixing the cells to coverslips, and staining for CD2, phosphotyrosine, and f-actin.

We found that CD2 is an essential co-receptor for NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). CD2 increased cytotoxicity when added to CD16 in an rADCC assay of primary NK cells against antibody-coated P815 cells (Fig 1). Similarly, CD2 knock-out cells (96% and 99% CD2 knock-out confirmed by flow cytometry) substantially inhibited ADCC towards the Raji B cell line with anti-CD38 mAb (daratumumab) in an ADCC assay (Fig 4). Additionally, costimulation with CD2 and CD16 resulted in significantly increased cell activation, as measured by phosphorylation of ERK, compared to CD2 and CD16 alone, suggesting CD2’s role as a costimulatory activation signal for CD16 (Fig 2). Furthermore, CD2 appears to have varying importance in natural cytotoxicity depending on the target cell line. CD2 knock-out did not affect the natural cytotoxicity toward pancreatic ductal adenocarcinoma (PDAC) cell lines (Fig 5) but substantially inhibited natural cytotoxicity towards the Raji B cell line (Fig 4). Lastly, we found polarization of CD2 toward the IS and corolla formation, suggesting that it plays a similar role in the assembly and organization of the IS in NK cells as in T cells (Fig 3). Overall, our data suggests that CD2 plays multiple roles in NK cell cytotoxicity, and thus it is most likely important for iPSC NK cells to express this receptor.

The role of CD2 as a co-receptor for primary NK cell ADCC is not surprising, given a previous study showing CD2 interaction with CD16 enhanced ADCC in adaptive NK cells. However, our research thus far is limited because only two cell lines have been tested, so further ADCC and rADCC assays should be performed to support our claim. Furthermore, the varying importance of CD2 in natural cytotoxicity bears further exploration. Further research should seek to understand this difference and test natural cytotoxicity against other cell lines. Lastly, although our data suggests CD2 is important in primary NK cells, this may not hold true for iSPC NK cells. Therefore, iSPC NK cells should be engineered to express CD2 and their cytotoxicity should be compared to iSPC NK cells without CD2.

Acknowledgements (Optional) :

Daniel Billadeau, Mayo Graduate School of Biomedical Sciences, Department of Immunology, and the SURF Program

References (Optional) :

1. Myers JA, Miller JS. Exploring the NK Cell Platform for Cancer Immunotherapy. Nature Reviews Clinical Oncology. 2020;18(2):85-100. doi:10.1038/s41571-020-0426-7

2. Ham H, Medlyn M, Billadeau DD. Locked and Loaded: Mechanisms Regulating Natural Killer Cell Lytic Granule Biogenesis and Release. Front Immunol. 2022;13:871106. Published 2022 Apr 26. doi:10.3389/fimmu.2022.871106

3. Binder C, Cvetkovski F, Sellberg F, et al. CD2 Immunoabiology. Front Immunol. 2020;11:1090. Published 2020 Jun 9. doi:10.3389/fimmu.2020.01090