UCLA

Natural Killer (NK) cells are part of the innate immune system, they respond to virally infected cells and to tumor formation. The NK cells are known to have a crucial role in mediating lysis against a variety of tumor cells through cell-mediated cytotoxicity or antibody-dependent cellular cytotoxicity (ADCC). Accumulated evidence from our lab has shown that anergized NK cells can induce resistance in transformed and healthy stem cells through secreted cytokines and cell-to-cell interaction. We previously showed that NK cells can significantly catalyze cytotoxicity against primary Oral Squamous Carcinoma Stem Cells (OSCSCs), human embryonic stem cells (hESCs), human mesenchymal stem cells (hMSCs), human dental pulp stem cells (hDPSCs), and induced human pluripotent stem cells (hiPSCs)[1, 2] whereas their differentiated cells lines were more resistant. In addition, our experimental data demonstrated that as the stem cells become significantly differentiated and significantly resistant to NK cytotoxicity when they were co-cultured in the presence of monocytes, probiotic bacteria and anti-IL10 monoclonal antibody. Thus, it is an emerging view in our laboratory that the stage of maturation and differentiation of healthy untransformed, as well as transformed tumorigenic, stem cells is predictive of their sensitivity to NK cells' mediated cytotoxicity[3]. We believe that NK cells have the ability to reduce inflammation and increase regeneration of new tissues by two ways: they lyse a small populations of stem cells that are incapable of differentiating and they support the best stem cells to maturation and differentiation after gaining cytokine producing function and up-regulate differentiation markers CD54, B7H1, and MHC Class 1 and down-regulate of stem cell marker, CD44.

We previously found that CD16 receptor on the NK cells induced split anergy in NK cells. Split anergy was coined by our lab to explain the phenomenon where NK cells lose cytotoxicity but gain the ability to secrete cytokines to support maturation of various healthy and transformed stem cell types. In this paper, our objective is to study probiotic bacteria because we found that they exacerbate the induction of split anergy in NK cells by triggering NK cells to secrete significant about of anti-inflammatory and pro-inflammatory cytokines, chemokines, and growth factors compared to CD16 receptor triggering of the NK cells alone. We also found that the combination of probiotic bacteria (sAJ2) plus monocytes and anti-IL10 monoclonal antibodies were the ultimate induction of split anergy in NK cells compared to probiotic bacteria, monocytes, or the antibody alone triggering the NK cells as together these monocyte, bacteria and antibodies have the great capacity to anergize NK cells to speed the processes of maturation and differentiation of various stem cell types and to speed the process of tissue regeneration and to reduce inflammation much more faster.

Transformed stem cells such as Oral Squamous Cancer Stem Cells (OSCSC), Pancreatic Carcinoma MIA PaCa-2, Human Lung Adenocarcinoma Epithelial Cell Line (A549), human Glioblastoma cell line (X02D) and Human Chronic Myelogenous Leukemia Cell line (K562) as well as non-transformed stem cell, Stem Cell of Apical Papilla (SCAP) were used in a standard 51Chromium release assay to determine their sensitivity or resistance against NK cell-mediated cytolysis. The secretion of key cytokines by NK cells induced by probiotic bacteria, such as Interferon-Gamma (IFN-γ), IL-10, and other cytokines, chemokines, and growth factors were determined using Enzyme-Linked Immunosorbent Assays (ELISAs). The death of tumor cells caused by different conditions of NK cell-treated probiotic bacteria were assessed through flow analysis using Propidium Iodide Stain. Transformed and non-transformed stem cells were differentiated using supernatants obtained from untreated NK cells, NK cells treated with IL-2 and anti-CD16mAb, and a combination of IL-2 plus anti-CD16mAb with our lab formulated probiotic bacteria, sonicated AJ2 (sAJ2) as well as other activators such as monocytes and anti-IL10 monoclonal antibody. We observed a universal step-wise differentiation level of stem cells when they were treated with supernatant of pre-conditioned NK cells that contained various level of IFN-γ production. The level of differentiation of the stem cells was confirmed through surface analysis using surface receptor markers CD54, CD44, B7H1, and MHC -1.

Our experimental findings showed that probiotic bacteria caused significant induction of split anergy in NK cells via significant secretion of many cytokines and lack of NK cells mediated cytotoxicity compared to IL-2 plus anti-CD16mAb treated NK cells (also known as conventional anergized NK). Along with the combination of monocytes and anti-IL10mAb significantly induced split anergy of the NK cells by speeding the differentiation and maturation process via secretion of significant amount of key cytokines, IFN-γ and TNF-α.