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Cystatin F, a cysteine peptidase inhibitor, is a potent modulator of NK cytotoxicity. By inhibiting granule-mediated cytotoxicity pathway, cystatin F induces formation of non-functional NK cell stage, called split-anergy. We show that N-glycosylation determines the localization and cellular function of cystatin F. Cystatin F mostly exhibited high-mannose glycosylation in U-937 cells, both high-mannose and complex glycosylation in NK-92 and primary NKs, and predominantly complex glycosylation in super-charged NKs. Manipulating N-glycosylation with kifunensine increased high-mannose glycosylation of cystatin F and lysosome localisation, which decreased cathepsin C activity and reduced NK cytotoxicity. Mannose-6-phosphate could significantly reduce the internalization of extracellular cystatin F. By comparing NK cells with different cytotoxic potentials, we found that high-mannose cystatin F was strongly associated with lysosomes and cathepsin C in NK-92 cell line. In contrast, in highly cytotoxic super-charged NKs, cystatin F with complex glycosylation was associated with the secretory pathway and less prone to inhibit cathepsin C. Modulating glycosylation to alter cystatin F localisation could increase the cytotoxicity of NK cells, thereby enhancing their therapeutic potential for treating cancer patients. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are functionally suppressed in the glioblastoma multiforme (GBM) tumor microenvironment. We have recently shown that survival and differentiation of cancer stem-like cells (CSCs)/poorly differentiated tumors are controlled through two distinct phenotypes of cytotoxic and non-cytotoxic/split anergized NK cells, respectively. In this paper, we studied the function of NK cells against brain CSCs/poorly differentiated GBM and their NK cell-differentiated counterparts. Brain CSCs/poorly differentiated GBM, differentiated by split anergized NK supernatants (supernatants from NK cells treated with IL-2 + anti-CD16mAb) expressed higher levels of CD54, B7H1 and MHC-I and were killed less by the NK cells, whereas their CSCs/poorly differentiated counterparts were highly susceptible to NK cell lysis. Resistance to NK cells and differentiation of brain CSCs/poorly differentiated GBM by split anergized NK cells were mediated by interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Brain CSCs/poorly differentiated GBM expressed low levels of TNFRs and IFN-γRs, and when differentiated and cultured with IL-2-treated NK cells, they induced increased secretion of pro-inflammatory cytokine interleukin (IL)-6 and chemokine IL-8 in the presence of decreased IFN-γ secretion. NK-induced differentiation of brain CSCs/poorly differentiated GBM cells was independent of the function of IL-6 and/or IL-8. The inability of NK cells to lyse GBM tumors and the presence of a sustained release of pro-inflammatory cytokines IL-6 and chemokine IL-8 in the presence of a decreased IFN-γ secretion may lead to the inadequacy of NK cells to differentiate GBM CSCs/poorly differentiated tumors, thus failing to control tumor growth. [ABSTRACT FROM AUTHOR]

The aim of this study is to identify the phenotype of resistant oral tumors, and to delineate the contribution of immune effectors to resistance of oral tumors. UCLA-1 oral tumors which were resistant to NK cell mediated cytotoxicity secreted increased amounts of IL-6, IL-1β, GM-CSF, and IL-8 when cultured with or without immune effectors. In addition, the levels of vascular endothelial growth factor (VEGF) secretion in the co-cultures of naïve immune effectors with UCLA-1 rose significantly when compared to tumor cells alone. IL-2 activated NK cells decreased VEGF secretion in all tumor cells. However, NK cells which were induced to undergo cell death with anti-CD16 antibody were not only unable to decrease VEGF secretion, but they also contributed further to the increase in VEGF secretion by oral tumors. Overall, we show in this paper that naïve as well as non-viable immune effectors may contribute to the growth and resistance of oral tumors by triggering the secretion of key tumor cell growth factors. [ABSTRACT FROM AUTHOR]

Cytotoxic T lymphocytes and natural killer (NK) cells kill target cells by two main mechanisms, namely, the perforin/granzymes and the Fas ligand (Fas-L) pathways. The preferential activation of either of these two mechanisms by target cells is not known. This study examined whether various NK stimuli regulate preferentially the perforin/granzyme or the Fas pathways during the NK-cell-mediated cytotoxic reaction (NK-CMC). Purified peripheral-blood-derived NK cells were stimulated with interleukin-2 (IL-2), IL-12, or interferon α (IFNα) and their response was analyzed by the reverse transcriptase/polymerase chain reaction (RT-PCR) for NK-associated gene expression and by the 51Cr-release assay for cytotoxic function. RT-PCR data revealed that the perforin, granzyme A and granzyme B mRNAs were constitutively expressed in unstimulated NK cells and the level of perforin mRNA was augmented following activation. IL-2 enhanced the level of Fas-L mRNA in NK cells; however, the Fas-L level was much lower than that obtained in activated T cells. NK-CMC against Fas-sensitive cells was examined in the presence of neutralizing anti-(Fas antigen receptor) (Fas-R) antibody (ZB-4) or EGTA/Mg2+, which inhibits the perforin/granzyme pathway but not the Fas Fas-L interaction. The human colon adenocarcinoma HT-29 cells were sensitized to anti-Fas-R antibody (CH-11) cytotoxicity following treatment with IFNγ. NK-CMC against untreated HT-29 cells was completely inhibited by EGTA/Mg2+ and was unaffected by ZB-4, while both EGTA/Mg2+ and ZB-4 partially inhibited NK-CMC against IFNγ-treated HT-29 cells. Similar findings to those obtained with untreated NK cells were observed with NK cells stimulated with IL-2, IL-2 plus IL-12 or IFNα. In contrast to IFNγ-treated HT-29 cells, the neutralizing anti-Fas antibody ZB-4 did not inhibit NK-CMC against Fas-sensitive U937, CEM or Jurkat tumor cells. These findings demonstrate that the Fas pathway is involved in NK-CMC against certain target cells but not all. Further, the data demonstrate that activation of NK cells by IL-2, IL-2 plus IL-12 or IFNα does not preferentially modulate the Fas-L-mediated killing by NK cells. [ABSTRACT FROM AUTHOR]