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Amyotrophic lateral sclerosis (ALS) is an auto-immune neurodegenerative disorder affecting the motor-neurons. The causes of ALS are heterogeneous, and are only partially understood to date. We studied percentage and function of immune cell subsets in particular natural killer (NK) and CD8+ T cells in an ALS patient and compared the results to those obtained from his genetically identical healthy twin in a longitudinal study. We found several basic mechanisms which were potentially involved in the disease induction and progression. Our findings demonstrate that ALS patient's peripheral blood contained higher NK and B cells and, lower T cell percentages compared with the healthy twin brother's peripheral blood. Significantly increased interferon-gamma secretion by anti-CD3/28 monoclonal antibody-treated peripheral blood mononuclear cells, and sorted CD8+ T cells were observed in the ALS patient, suggesting that hyper-responsiveness of T cell compartment could be a potential mechanism of ALS progression. Significant increase in NK cell function due to genetic mutations in ALS associated genes may partly be responsible for the increase expansion and function of CD8+ T cells with effector/memory phenotype, in addition to direct activation and expansion of antigen specific T cells by such mutations. Weekly N-acetyl cysteine infusion to block cell death in patient in addition to a number of other therapies listed in this paper were not effective, and even though the treatments might have extended the patient's life, it was not curative. Therefore, activated CD8+ T and NK cells are likely cells targeting motor neurons in the patient, and strategies should be designed to decrease the aggressive nature of these cells to achieve longer lasting therapeutic benefits.

Amyotrophic lateral sclerosis (ALS) is a neurological disease characterized by the progressive loss of motor neurons in the brain and spinal cord. No effective therapeutic strategies have been established thus far, and therefore there is a significant unmet need for effective therapeutics to arrest the disease and reverse the pathologies induced by it. Although the cause of ALS is not well-defined, it appears to be heterogenous. Currently over 20 genes have been found to be associated with ALS. Family history can only be found in 10% of ALS patients, but in the remaining 90% no association with family history is found. The most common genetic causes are expansion in the C9orf72 gene and mutations in superoxide dismutase 1, TDP-43, and FUS. In our recent study, we also found mutations in TDP43 and FUS in ALS patients. To understand the pathogenesis of the disease, we set ourselves the task of analyzing the phenotype and function of all key immune effectors in ALS patients, comparing them with either a genetically healthy twin or healthy individuals. Our study demonstrated a significant increase in functional activation of NK and CD8+ T cytotoxic immune effectors and release of significant IFN-γ not only by the effector cells but also in the serum of ALS patients. Longitudinal analysis of CD8+ T cell-mediated IFN-γ secretion from ALS patients demonstrated continued and sustained increase in IFN-γ secretion with periods of decrease which coincided with certain treatments; however, the effects were largely short-lived. N-acetyl cysteine (NAC), one of the treatments used, is known to block cell death; however, even though such treatment was able to block most of the proinflammatory cytokines, chemokines, and growth factor release, it was not able to block IFN-γ and TNF-α, the two cytokines we had demonstrated previously to induce differentiation of the cells. In this review, we discuss the contribution of cytotoxic effector cells, especially primary NK cells, supercharged NK cells (sNK), and the contribution of sNK cells in expansion and functional activation of CD8+ T cells to memory/effector T cells in the pathogenesis of ALS. Potential new targeted therapeutic strategies are also discussed.

Our recent studies indicated that amyotrophic lateral sclerosis (ALS) patients suffer from significantly elevated levels of interferon-gamma (IFN-γ) secretion by natural killer (NK) and CD8+ T cells, which may be responsible for the immune-pathologies seen in central nervous system and in peripheral organs of the patients. In order to counter such elevated induction of IFN-γ in patients we designed a treatment strategy to increase anti-inflammatory cytokine interleukin-10 (IL-10) by the use of probiotic strains which significantly increase the levels of IL-10. Therefore, in this paper we demonstrate disease specific functions of Al-Pro (AJ3) formulated for the adjunct treatment of auto-immune diseases including ALS, and compared the function with CA/I-Pro (AJ4) for the treatment of cancer and viral diseases, and NK-CLK (AJ2) for maintenance of immune balance and promotion of disease prevention. The three different formulations of probiotic bacteria have distinct profiles of activation of peripheral blood mononuclear cells (PBMCs), NK, and CD8+ T cells, and their induced activation is different from those mediated by either IL-2 or IL-2 + anti-CD16 monoclonal antibodies (mAbs) or IL-2 + anti-CD3/CD28 mAbs. IL-2 + anti-CD16 mAb activation of PBMCs and NK cells had the highest IFN-γ/IL-10 ratio, whereas IL-2 combination with sAJ4 had the next highest followed by IL-2 + sAJ2 and the lowest was seen with IL-2 + sAJ3. Accordingly, the highest secretion of IFN-γ was seen when the PBMCs and NK cells were treated with IL-2 + sAJ4, intermediate for IL-2 + sAJ2 and the lowest with IL-2 + sAJ3. The levels of IFN-γ induction and the ratio of IFN-γ to IL-10 induced by different probiotic bacteria formulation in the absence of IL-2 treatment remained much lower when compared to those treated in the presence of IL-2. Of note is the difference between NK cells and CD8+ T cells in which synergistic induction of IFN-y by IL-2 + sAJ4 was significantly higher in NK cells than those seen by CD8+ T cells. Based on these results, sAJ3 should be effective in alleviating auto-immunity seen in ALS since it will greatly regulate the levels and function of IFN-γ negatively, decreasing overactivation of cytotoxic immune effectors and prevention of death in motor neurons.

Programmed cell death-1 (PD-1) immunoinhibitory receptor expression is found on T cells, B cells, natural killer (NK) cells, and myeloid cells. Upon activation of T cells through peptide-major histocompatibility complex (MHC) engagement of the T cell receptor and costimulatory signaling, checkpoints including PD-1 are activated to regulate T cells. Since decreased expression of PD-1 in mice model was found to be associated with breakdown of peripheral tolerance, and demonstrated autoimmune disease characteristic, this receptor may be important therapeutic target for autoimmunity. In addition, decreased NK cell numbers and cytotoxicity in peripheral blood and altered expression of activating receptors and cytokine secretion of NK cells was seen in autoimmune disease patients. Therefore, in this review we discuss the relevance of PD-1 function in NK and T cells in autoimmunity, and demonstrate similarities and differences of its function in autoimmune diseases and cancer. Thus, PD-1 can be targeted to treat each disease entity accordingly. In cancer, the function of PD-1 can be blocked in order to enhance immune activation, whereas in autoimmune diseases it can be enhanced to block heightened immune function. However, we are far from understanding the exact functioning of this receptor in a complex tissue microenvironment, and further studies are required to establish its function at different stages of the disease, and at different stages of the maturation of immune effectors.

Elucidating the role of probiotic bacteria in health and disease perhaps constitutes one of the most exciting and fastest growing fields in medicine as we uncover the beneficial roles of these bacteria in many disease processes including cancer. We and others have reported previously that probiotic bacteria play a significant role in the activation of many cells including the cancer fighting natural killer (NK) cells. NK cells are the key immune effectors which control tumor growth and metastasis due to their ability to mediate direct cytotoxicity and/or differentiation of cancer stem cells/undifferentiated tumors through secreted and membrane bound interferon-gamma and tumor necrosis factor-alpha. In this review, we present an overview of recent studies from our laboratory and those of the others on their beneficial effects on immune cell function in particular on NK cells. In addition, we also highlight the current understanding of the role of probiotics in enhancement of the effectiveness of cancer therapeutics. Moreover, we discuss the functional impairment of cancer patients' NK cells and the role of probiotics in reversal of such functional impairment. NK cell-based immuno-therapies in combination with well-selected strains of probiotic bacteria may probably represent one of the best adjunct therapeutic approaches to prevent and treat cancer in the future.

Osteoclasts (OCs) and much less dendritic cells (DCs) induce significant expansion and functional activation of NK cells, and furthermore, the OC-expanded NK cells preferentially increase the expansion and activation of CD8+ T cells by targeting CD4+ T cells. When autologous OCs were used to expand patient NK cells much lower percentages of expanded CD8+ T cells, decreased numbers of expanded NK cells and decreased functions of NK cells could be observed, and the addition of allogeneic healthy OCs increased the patients’ NK function. Mechanistically, OC-expanded NK cells were found to lyse CD4+ T cells but not CD8+ T cells suggesting potential selection of CD8+ T cells before their expansion by OC activated NK cells. In agreement, Increased IFN-γ secretion, and NK cell-mediated cytotoxicity and higher percentages of CD8+ T cells, in various tissue compartments of oral tumor-bearing hu-BLT mice in response to immunotherapy by OC-expanded NK cells were observed. Thus, our results indicate an important relationship between NK and CD8+ T cells.

Background The cholinesterase inhibitor therapeutics (CI) approved for use in Alzheimer's disease (AD) are palliative for a limited time. Objective To examine the outcome of AD patients with add-on therapy of the omega-3 fatty acid drink Smartfish. Methods We performed a prospective study using Mini-Mental State Examination, amyloid-β (Aβ) phagocytosis blood assay, and RNA-seq of peripheral blood mononuclear cells in 28 neurodegenerative patients who had failed their therapies, including 8 subjective cognitive impairment (SCI), 8 mild cognitive impairment (MCI), 2 AD dementia, 1 frontotemporal dementia (FTD), 2 vascular cognitive impairment, and 3 dementia with Lewy bodies (DLB) patients. Results MCI, FTD, and DLB patients patients volunteered for the addition of a ω-3 fatty acid drink Smartfish protected by anti-oxidants to failing CI therapy. On this therapy, all MCI patients improved in the first year energy transcripts, Aβ phagocytosis, cognition, and activities of daily living; in the long term, they remained in MCI status two to 4.5 years. All FTD and DLB patients rapidly progressed to dementia. On in vivo or in vitroω-3 treatments, peripheral blood mononuclear cells of MCI patients upregulated energy enzymes for glycolysis and citric acid cycle, as well as the anti-inflammatory circadian genes CLOCK and ARNTL2. Conclusion Add-on ω-3 therapy to CI may delay dementia in certain patients who had failed single CI therapy.

Natural killer (NK) cells are the key immune effectors with the ability to mediate selection and differentiation of a number of different cancer stem cells/undifferentiated tumors via lysis, and secreted or membrane-bound interferon (IFN)-γ and tumor necrosis factor (TNF)-α, respectively, leading to curtailment of tumor growth and metastasis. In this review, we present an overview of our recent findings on the biology and significance of NK cells in selection and differentiation of stem-like tumors using in vitro and in vivo studies conducted in humanized-BLT mice and in cancer patients. In addition, we present current advances in NK cell expansion and therapeutic delivery, and discuss the utility of allogeneic supercharged NK cells in the treatment of cancer patients. Moreover, we discuss the potential loss of NK cell numbers and function at the neoplastic and pre-neoplastic stages of tumorigenesis in induction and progression of pancreatic cancer. Therefore, because of their indispensable role in targeting cancer stem-like/undifferentiated tumors, NK cells should be placed high in the armamentarium of tumor immunotherapy. A combination of allogeneic supercharged NK cells with other immunotherapeutic strategies such as oncolytic viruses, antibody-dependent cellular cytotoxicity (ADCC)-inducing antibodies, checkpoint inhibitors, chimeric antigen receptor (CAR) T cells, CAR NK cells, and chemotherapeutic and radiotherapeutic strategies can be used for the ultimate goal of tumor eradication. Keywords: NK cells, cancer stem cells, CSCs, differentiation, cancer immunotherapy, osteoclasts, supercharged NK cells, humanized-BLT mice, pre-neoplastic, KRAS

We have previously demonstrated that natural killer (NK) cells are the main immune effectors that can mediate selection and differentiation of different cancer stem cells and undifferentiated tumors via lysis and secreted or membrane-bound interferon-γ and tumor necrosis factor-α, respectively. This leads to growth inhibition and tumor metastasis curtailment. In this review, we present an overview of our findings on NK cell biology and its significance in selection and differentiation of stem-like tumors using in vitro and in vivo studies conducted in nonobese diabetic/severe combined immunodeficiency (scid)/interleukin-Rγ--, humanized-bone-marrow/liver/thymus (hu-BLT) mice, and those of human cancer patients. Moreover, we present recent advances in NK cell expansion and therapeutic delivery and discuss the superiority of allogeneic supercharged NK cells over their autologous counterparts for cancer treatment. We review potential loss of NK cell numbers and function at neoplastic and preneoplastic stages of tumorigenesis as a potential mechanism for pancreatic cancer induction and progression. We believe that NK cells should be placed highly in the armamentarium of tumor immunotherapy due to their indispensable role in targeting cancer stem-like/poorly differentiated tumors and a variety of other key NK cell functions that are discussed in this report, including their role in CD8+ T-cell expansion and targeting gene knockout or dedifferentiated tumors. The combination of allogeneic supercharged NK cells and other immunotherapeutic strategies such as oncolytic viruses, antibody-dependent cellular cytotoxicity-inducing antibodies, checkpoint inhibitors, chimeric antigen receptor (CAR)-T cells and CAR-NK cells, chemotherapeutics, and radiotherapeutic strategies can be used for optimal eradication of tumors.

We investigated the use of a system with an ingestible sensor (Proteus Digital Health Feedback system) coencapsulated with antiretrovirals (ARVs) to measure real-time adherence. To assess the safety and impact, if any, coencapsulation might have on ARV concentrations, we evaluated the pharmacokinetics of ARVs coencapsulated with an ingestible sensor for eight commonly used fixed-dose combination ARVs: emtricitabine (FTC)/tenofovir disoproxil fumarate (TDF); FTC/tenofovir alafenamide (TAF); efavirenz (EFV)/FTC/TDF; abacavir (ABC)/lamivudine (3TC); dolutegravir (DTG)/ABC/3TC; rilpivirine (RPV)/TAF/FTC; elvitegravir (EVG)/cobicistat (COBI)/FTC/TAF; and bictegravir (BIC)/FTC/TAF. The steady-state apparent peak plasma concentration (Cmax) and area under the concentration-time curve (AUC) were determined from plasma concentrations measured at predose, 1, 2, 4, and 6 h postdose, and compared with literature values. A total of 49 unique patients on stable regimens for at least 12 weeks with undetectable viral loads were recruited. Cmax and AUC values were not statistically significantly different from literature values for all of the formulations except the Cmax of FTC/TDF, Cmax of BIC, and the Cmax of RPV. In a subsequent evaluation of FTC/TDF and BIC/FTC/TAF using a crossover design, the geometric mean ratio (GMR) between the coencapsulated and the unencapsulated formulations for FTC/TDF were the following: FTC, 84.6% (90% confidence interval [CI] 66.6–107.4) for AUC and 77.5% (60.1–99.9) for Cmax. For tenofovir (TFV), the GMR was 96.2% (90% CI 89.2–103.8) for AUC and 87.3% (64.2–118.7) for Cmax. The GMR for BIC (from the BIC/FTC/TAF formulation) was 98.0% (90% CI 84.5–113.5) for AUC and 89.9% (84.5–95.7) for Cmax. The observed deviation in FTC/TDF (Truvada) may be due to participant characteristics, fasted/fed conditions, and/or random variation and may warrant further investigations with a larger sample size. These findings provide assurance for use of coencapsulated ARVs for future HIV treatment-adherence research.

Glioblastomas remain the most lethal primary brain tumors. Natural killer (NK) cell-based therapy is a promising immunotherapeutic strategy in the treatment of glioblastomas, since these cells can select and lyse therapy-resistant glioblastoma stem-like cells (GSLCs). Immunotherapy with super-charged NK cells has a potential as antitumor approach since we found their efficiency to kill patient-derived GSLCs in 2D and 3D models, potentially reversing the immunosuppression also seen in the patients. In addition to their potent cytotoxicity, NK cells secrete IFN-γ, upregulate GSLC surface expression of CD54 and MHC class I and increase sensitivity of GSLCs to chemotherapeutic drugs. Moreover, NK cell localization in peri-vascular regions in glioblastoma tissues and their close contact with GSLCs in tumorospheres suggests their ability to infiltrate glioblastoma tumors and target GSLCs. Due to GSLC heterogeneity and plasticity in regards to their stage of differentiation personalized immunotherapeutic strategies should be designed to effectively target glioblastomas.

The majority of the previous reports on NK cells use cross-sectional studies to establish the status of patient NK cell function, however such studies fail to evaluate the immune status of the patients on a continuous basis from the disease-free stage to progression of cancer. In this study, we performed a prospective study of the immune function by continuously monitoring the NK numbers, expansion and function of a pancreatic cancer patient from 1/6/2016 to 2/14/2019. The results indicated that at initial stages of the disease where no overt disease was identified, the patient had consistently higher percentages of NK and B cells and lower percentages of CD3 + T cells in the peripheral blood. The percentages of CD14 + monocytes were similar at the initial stages of the disease, and at the later stages of the disease, it increased and remained higher in the patient when compared to those from healthy donors. The numbers of expanded NK cells and the cytotoxic function, as well as secretion of IFN-γ from primary and osteoclast expanded patient NK cells remained consistently low throughout the years of follow up. Similarly, the majority of cytokines in patient’s serum remained lower with the exception of IL-6 which was higher. The IFN-γ secreted from the patients’ NK cells had much lower ability to differentiate the poorly differentiated oral tumors as assessed by their lack of ability to upregulate differentiation antigens. Overall, before any evidence of overt disease, patient NK cells exhibited significant dysfunction. Intervention at the stage of no disease or minimal disease may be important for the prevention of pancreatic cancer progression.

We have recently shown that natural killer (NK) cells select and differentiate cancer stem cells (CSCs)/undifferentiated tumors via secreted and membrane bound IFN-gamma (IFN-γ) and TNF-alpha (TNF-α), preventing tumor growth and inducing remodeling of the tumor microenvironment. Since many conventional therapeutic strategies, including chemotherapy and radiotherapy remain fairly unsuccessful in treating CSCs/poorly differentiated tumors, there has been an increasing interest in NK cell-targeted immunotherapy for the treatment of aggressive tumors. In our recent studies, we used humanized-BLT (hu-BLT) mouse model with transplanted human bone marrow, liver and thymus to demonstrate the efficacy of adoptive transfer of ex vivo expanded, super-charged NK cells in selection and differentiation of stem-like tumors within the context of a fully reconstituted human immune system. Furthermore, we have demonstrated that CSCs differentiated with split-anergized NK cells prior to implantation in hu-BLT mice were not able to grow or metastasize. However, when NK cell-mediated tumor differentiation was blocked by the addition of antibodies to IFN-γ and TNF-α, tumors grew and metastasized. In this review, we present current advances in NK cell expansion and therapeutic delivery, and discuss the utility of allogeneic super-charged NK cells in treatment of cancer patients. In addition, NK suppression occurs not only at the stage of overt cancer, but also at the pre-neoplastic stage. Therefore, due to the indispensable role of NK cells in targeting CSCs/undifferentiated tumors and their role in differentiation of the tumors, NK cells should be placed high in the armamentarium of tumor immunotherapy.

Natural killer (NK) cells are the main immune effectors with the ability to mediate selection and differentiation of a number of different cancer stem cells/undifferentiated or poorly differentiated tumors via lysis, and secreted or membrane bound IFN-γ and TNF-α. Tumor differentiation by NK cells leads to growth inhibition and curtailment of tumor metastasis. In this chapter, we present an overview of our recent and past findings on the biology and significance of NK cells in the selection/differentiation of stem-like tumors using in vitro and in vivo studies conducted in the NSG and humanized-BLT mice and those of cancer patients. In addition, we discuss the significance of heightened NK cell function in a great variety of gene knockout mice, providing a potential rationale for the mechanisms of the activation of NK cells in these animals. Moreover, we report on the recent advances in NK cell expansion and therapeutic delivery, and discuss the superiority of allogeneic supercharged NK cells over their autologous counterparts in the treatment of cancer patients. Furthermore, we discuss the potential loss of NK cell numbers and function at the neoplastic and preneoplastic stages of tumorigenesis as potential mechanisms for the induction and progression of pancreatic cancer. We also report on the novel ways of assessments of NK function within the 3D model of tumorospheres, to better represent the tumor microenvironment. Therefore, because of their indispensable role in targeting cancer stem-like/undifferentiated tumors, and a variety of other key functions of NK cells reviewed in this report, including their role in the selection and expansion of CD8 + T cells, and their potential functional inactivation by the tumors within the tumor microenvironment, which are mitigated by the mode of activation and expansion of NK cells, these cells should be placed high in the armamentarium of tumor immunotherapy. The combination of allogeneic supercharged NK cells with other therapeutic strategies such as the use of oncolytic viruses, ADCC-inducing antibodies, checkpoint inhibitors, CAR-T, CAR-NK, chemotherapeutic, and radiotherapeutic strategies can be used for the optimal treatment strategies.

Chronic lymphocytic leukemia (CLL) accounts for 10% of hematologic malignancies. CLL is a malignancy of CD5+ B cells and it is characterized by the accumulation of small, mature-appearing neoplastic lymphocytes in the blood, bone marrow, and secondary lymphoid tissues. In the present case, a middle-aged female patient with poor prognosis unmutated IGHV CLL achieved cytogenetic and molecular remission with minimal adverse events following six cycles of low dose recombinant human IL-2 (rIL-2) in combination with low dose targeted venetoclax. Personalized low dose rIL-2 in combination with either lenalidomide or venetoclax mediates natural killer stimulation and is an effective non-toxic immunotherapy administered in the outpatient setting for poor prognosis CLL.

Natural killer (NK) cells are known to select and differentiate cancer stem-like cells/undifferentiated tumors via lysis, and secreted/membrane bound IFN-γ and TNF-α respectively, resulting in the control of tumor growth. Several in vivo mouse models including humanized-BLT mice have been used to study the biology and significance of NK cells in selection/differentiation of stem-like tumors within the context of a reconstituted human immune system. In addition, we discuss the evidence and significance of NK cell loss at the pre-neoplastic stage. Therefore, because of their indispensable role in targeting CSCs/undifferentiated tumors, NK-cells should be placed high in the armamentarium of tumor therapy.

Background and Aims: We have previously demonstrated that the stage of differentiation of tumors has profound effect on the function of NK cells, and that stem-like/poorly differentiated tumors were preferentially targeted by the NK cells. Therefore, in this study we determined the role of super-charged NK cells in immune mobilization, lysis, and differentiation of stem-like/undifferentiated tumors implanted in the pancreas of humanized-BLT (hu-BLT) mice fed with or without AJ2 probiotics. The phenotype, growth rate and metastatic potential of pancreatic tumors differentiated by the NK cells (NK-differentiated) or patient derived differentiated or stem-like/undifferentiated pancreatic tumors were investigated. Methods: Pancreatic tumor implantation was performed in NSG and hu-BLT mice. Stage of differentiation of tumors was determined using our published criteria for well-differentiated tumors exhibiting higher surface expression of MHC- class I, CD54, and PD-L1 (B7H1) and lower expression of CD44 receptors. The inverse was seen for poorly-differentiated tumors. Results: Stem-like/undifferentiated pancreatic tumors grew rapidly and formed large tumors and exhibited lower expression of above-mentioned differentiation antigens in the pancreas of NSG and hu-BLT mice. Unlike stem-like/undifferentiated tumors, NK-differentiated MP2 (MiaPaCa-2) tumors or patient-derived differentiated tumors were not able to grow or grew smaller tumors, and were unable to metastasize in NSG or hu-BLT mice, and they were susceptible to chemotherapeutic drugs. Stem-like/undifferentiated pancreatic tumors implanted in the pancreas of hu-BLT mice and injected with super-charged NK cells formed much smaller tumors, proliferated less, and exhibited differentiated phenotype. When differentiation of stem-like tumors by the NK cells was prevented by the addition of antibodies to IFN-&gamma, and TNF-&alpha, tumors grew rapidly and metastasized, and they remained resistant to chemotherapeutic drugs. Greater numbers of immune cells infiltrated the tumors of NK-injected and AJ2-probiotic bacteria-fed mice. Moreover, increased IFN-&gamma, secretion in the presence of decreased IL-6 was seen in tumors resected and cultured from NK-injected and AJ2 fed mice. Tumor-induced decreases in NK cytotoxicity and IFN-&gamma, secretion were restored/increased within PBMCs, spleen, and bone marrow when mice received NK cells and were fed with AJ2. Conclusion: NK cells prevent growth of pancreatic tumors through lysis and differentiation, thereby curtailing the growth and metastatic potential of stem-like/undifferentiated-tumors.

Therapeutic role of NK cells in solid tumors was challenged previously even though their role in hematological malignancies has clearly been established. Furthermore, functions and numbers of NK cells are greatly suppressed in oral cancer patients necessitating effective future NK immunotherapeutic strategies to aid in the control of disease. The humanized-BLT (hu-BLT) mice were used to implant stem-like/undifferentiated oral tumors to study the role of super-charged NK cells with and without feeding with AJ2 probiotic bacteria. Implanted CSC/undifferentiated tumors resected from NK-injected mice exhibited differentiated phenotype, grew slowly, and did not cause weight loss, whereas those from tumor-bearing mice without NK-injection remained relatively more stem-like/poorly-differentiated, grew faster, and caused significant weight loss. Moreover, in vitro NK-differentiated tumors were sensitive to chemotherapeutic drugs, and when implanted in the oral-cavity grew no or very small tumors in mice. When NK-mediated differentiation of tumors was blocked by IFN-γ and TNF-αantibodies before implantation, tumors grew rapidly, remained stem-like/poorly-differentiated and became resistant to chemotherapeutic drugs. Loss of NK cytotoxicity and decreased IFN-γ secretion in tumor-bearing mice in PBMCs, splenocytes, bone marrow derived immune cells and enriched NK cells was restored by the injection of super-charged NK cells with or without feeding with AJ2. Much greater infiltration of CD45+ and T cells were observed in tumors resected from the mice, along with the restored secretion of IFN-γ from purified T cells from splenocytes in NK-injected tumor-bearing mice fed with AJ2 probiotic bacteria. Thus, super-charged NK cells prevent tumor growth by restoring effector function resulting in differentiation of CSCs/undifferentiated-tumors in hu-BLT mice.