Abstract
Simple SummaryNatural Killer cells are known to eliminate tumors directly or via antibody dependent cellular cytotoxicity. The complete modes and mechanisms of such killings are yet to be delineated. It is also unclear at what stages of tumor differentiation NK cells are capable of mediating the two modes of tumor killing. In this report we provide evidence that NK cells mediate killing of both stem-like/poorly differentiated tumors and well-differentiated tumors via direct cytotoxicity and antibody dependent cellular cytotoxicity, respectively. By using antibodies to MICA/B, EGFR and PDL1 surface receptors expressed on well-differentiated but not on stem-like/poorly differentiated tumors we demonstrate significant NK cell mediated antibody dependent cellular cytotoxicity in the absence of direct killing. In addition, our results suggested the possibility of CD16 receptors mediating both direct cytotoxicity and antibody dependent cellular cytotoxicity, resulting in the competitive use of these receptors in either direct killing or antibody dependent cellular cytotoxicity.Tumor cells are known to upregulate major histocompatibility complex-class I chain related proteins A and B (MICA/B) expression under stress conditions or due to radiation exposure. However, it is not clear whether there are specific stages of cellular maturation in which these ligands are upregulated or whether the natural killer (NK) cells differentially target these tumors in direct cytotoxicity or antibody-dependent cell cytotoxicity (ADCC). We used freshly isolated primary and osteoclast (OCs)-expanded NK cells to determine the degree of direct cytotoxicity or of ADCC using anti-MICA/B monoclonal antibodies (mAbs) against oral stem-like/poorly-differentiated oral squamous cancer stem cells (OSCSCs) and Mia PaCa-2 (MP2) pancreatic tumors as well as their well-differentiated counterparts: namely, oral squamous carcinoma cells (OSCCs) and pancreatic PL12 tumors. By using phenotypic and functional analysis, we demonstrated that OSCSCs and MP2 tumors were primary targets of direct cytotoxicity by freshly isolated NK cells and not by ADCC mediated by anti-MICA/B mAbs, which was likely due to the lower surface expression of MICA/B. However, the inverse was seen when their MICA/B-expressing differentiated counterparts, OSCCs and PL12 tumors, were used in direct cytotoxicity and ADCC, in which there was lower direct cytotoxicity but higher ADCC mediated by the NK cells. Differentiation of the OSCSCs and MP2 tumors by NK cell-supernatants abolished the direct killing of these tumors by the NK cells while enhancing NK cell-mediated ADCC due to the increased expression of MICA/B on the surface of these tumors. We further report that both direct killing and ADCC against MICA/B expressing tumors were significantly diminished by cancer patients’ NK cells. Surprisingly, OC-expanded NK cells, unlike primary interleukin-2 (IL-2) activated NK cells, were found to kill OSCCs and PL12 tumors, and under these conditions, we did not observe significant ADCC using anti-MICA/B mAbs, even though the tumors expressed a higher surface expression of MICA/B. In addition, differentiated tumor cells also expressed higher levels of surface epidermal growth factor receptor (EGFR) and programmed death-ligand 1(PDL1) and were more susceptible to NK cell-mediated ADCC in the presence of anti-EGFR and anti-PDL1 mAbs compared to their stem-like/poorly differentiated counterparts. Overall, these results suggested the possibility of CD16 receptors mediating both direct cytotoxicity and ADCC, resulting in the competitive use of these receptors in either direct killing or ADCC, depending on the differentiation status of tumor cells and the stage of maturation and activation of NK cells.
Highlights
Natural killer (NK) cells were first discovered as a functional cell type in 1970 and were named by Kiessling et al in 1975 [1]
We detected a lower expression of Nkp44, CD94, NKG2D, and KIR2, and a higher expression of Nkp30, Nkp46, and KIR3 on CD16+ NK cells from cancer patients (Figure 1I–K and Figure S3)
We have previously shown that osteoclast (OC)-expanded NK cells exhibit a greater potential to directly kill tumor cells while expressing lower levels of CD16 receptors when compared to freshly isolated primary NK cells [61]
Summary
Natural killer (NK) cells were first discovered as a functional cell type in 1970 and were named by Kiessling et al in 1975 [1]. NK cells mediate their functions through several important activating and inhibitory cell receptors such as CD16, NKG2D, natural cytotoxicity receptors (NCR), killer immunoglobulin-like receptors (KIR), and the NKG2 family of receptors, which form heterodimers with CD94 [5,6,7]. The balance between activating and inhibitory signals that NK cells receive through the surface receptors determines their functional fate [5]. As such, activated NK cells are able to recognize and lyse tumor cells expressing certain surface receptors without prior antigenic sensitization [8,9]. Differentiated tumors were found to have higher expression of epidermal growth factor receptor (EGFR) [15,16] and programmed death-ligand 1(PDL1) [17,18]
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