Umbilical Cord Blood T Cells Express the Natural Cytotoxicity Receptors, NKp30, NKp44 and NKp46 after IL-15 Stimulation, but Only NKp30 Is Functional.
Authors: Tang Qin , Grzywacz Bartosz , Wang Hongbo , Kataria Nandini , Wagner John E. , Blazar Bruce R. , Miller Jeffrey S. , Verneris Michael R. 
Published: October 2019

Abstract
The natural cytotoxicity receptors (NCRs), NKp30, NKp44 and NKp46, are a recently identified group of receptors that play a central role in NK cell killing of malignant cells. Previous studies show that the NCRs are restricted to the NK cell lineage. However, we noted that a small fraction of freshly isolated umbilical cord blood (UCB) T cells expressed NKp30 (1.7±0.9%, n=8), but not NKp44 (0.4±0.4%, n=8) or NKp46 (0.5±0.4%, n=8). Thus, we investigated whether NCRs could be induced on either UCB or adult peripheral blood (PB) T cells after culture in IL-15 for 14 days. This cytokine was chosen since previous studies show that IL-15 induces other NK cell receptors on both UCB and PB T cells. Surprisingly, UCB, but not PB T cells, acquired NKp30 (37±10% vs 4±2%, p=0.01), NKp44 (41±20% vs. 1±0.6%, p=0.01) and NKp46 (13±7% vs 0.6±0.5%, p=0.01). NCRs were found mainly on CD8+ or CD3+CD56+ UCB T cells. Further studies addressed whether other cytokines could induce NCR expression on UCB T cells. Both IL-2 and IL-15 showed a dose dependent induction of NCRs. In contrast, IL-7 induced only NKp30 on UCB T cells, but in a non-dose dependent manner. IL-4 abrogated NCR expression, even in the presence of IL-15. Considering that the ligands for the NCRs are not yet elucidated, we performed functional studies using agonist mAbs and NK cells served as controls. Surprisingly, while all three NCRs were expressed on IL-15 expanded UCB T cells, only NKp30 was functional as demonstrated by degranulation (CD107a), IFN-γ release, and redirected killing assays (reverse ADCC). Previous studies show that the NKp30, −44 and −46 cooperate to induce NK killing. However, the simultaneous triggering of all 3 NCRs did not increase UCB T cell cytotoxicity. Some NK cell receptors modulate TCR triggering, but this was not the case with NCRs. We did find that another NK cell receptor, NKG2D, enhanced the cytoxic triggering of NKp30, but not other NCRs. To address the lack of function of NKp44 and −46 on UCB T cells, the expression of adapter proteins required for signaling through these receptors were determined. The proximal receptor for NKp44 signaling, DAP12, was absent in UCB T cells. Thus, the lack of DAP12 likely explains the absence of NKp44 signaling. Both of the adapters used by NKp30 and −46 (FcεR1γ and CD3ζ) were detected. As stated above, NKp46 was expressed on significantly fewer cells than NKp30 (13±7% vs 37±10%) and this likely accounts for the lack of NKp46 function. Considering that UCB contains mostly naive T cells, we hypothesized that PB naive T cells may also acquire NCRs. To test this, PB T cell subsets (naive, central memory and effector memory) were FACS sorted and cultured for 14 days in IL-15. NKp30 was induced on a small number of naive, but not memory PB T cells. Unlike UCB T cells, NKp30 on naive PB T cells was non-functional. PB T cells lacked FcεR1γ, while UCB T cells expressed it, suggesting that this adapter protein is critical for NKp30 signaling. Collectively, these results show that UCB T cells are unique in their ability to acquire NCRs. Moreover, only NKp30 is functional. The lack of function of NKp44 and -46 is due to the absence of DAP12 and the low level of NKp46 expression, respectively. Lastly, while some naive PB T cells can acquire NKp30, it is not functional due to the lack of FcεR1γ. Such studies highlight the differences between UCB and PB T cells and challenge the dogma that NCRs are NK cell specific.