Blocking LLT1-CD161 interaction enhances natural killer cell-mediated lysis of triple-negative breast cancer cells

Purpose: Triple-negative breast cancer (TNBC) accounts for 20 percent of all breast cancer cases and is known to be the most invasive form of breast cancer. TNBC’s absence of estrogen, progesterone, and human epidermal growth factor-2 receptors makes utilizing hormonal treatments ineffective in suppressing tumor growth. TNBC is associated with poorer prognosis and higher incidences of relapse. Therefore, natural killer cell-mediated immunotherapy shows potential as a treatment option for TNBC. Natural killer cells (NK) are innate lymphoid cells that serves its role in the immune system to eradicate infected and tumor cells. NK cell function is regulated through its receptors interacting with activating and inhibitory ligands on target cells. Lectin-like Transcript-1 (LLT1, CLEC2D) is a counter-receptor that interacts with CD161 (NKRP1A) and inhibits NK cell activation. Our study demonstrated that by blocking TNBC’s LLT1 interaction with CD161 with antibodies increases lysis of TNBCs by NK cells. Methods: We have identified the expression and function of LLT1 on TNBC cell lines MDA-MB-231 and MDA-MB-436 by flow cytometry, western blot, immunofluorescent microscopy, and chromium-release assay. LLT1 expression at the cell surface was decreased through gene knockdown with small interference RNA (siRNA) transfection. Primary NK cells were isolated from peripheral blood mononuclear cells from healthy individuals and then were co-incubated with chromium-labeled TNBCs for quantification of specific lysis of TNBCs by NK cells. Results: Our results have demonstrated a higher expression of LLT1 on TNBCs than non-tumorigenic breast cell line MCF10A. We have shown that blocking LLT1 interaction with CD161 with antibodies on TNBCs have increased lysis of TNBCs by primary NK cells. We have also shown that gene knockdown of LLT1 decreases cell surface expression of LLT1 on TNBCs and increases lysis of TNBCs by NK cells. Conclusions: LLT1 expressed on TNBCs is a ligand that interacts with NK receptor CD161 and sends an inhibitory signal to the NK cell thus serving its role for TNBCs to evade immunosurveillance. Respectively, blocking LLT1 with antibodies on TNBCs and decreasing expression of LLT1 by gene knockdown increases susceptibility of TNBCs to NK cell-mediated lysis. Blocking interaction between LLT1 and CD161 with antibodies activates lysis by NK cells and will open a possible new immunotherapeutic strategy for patients diagnosed with TNBC.