Natural killer (NK) cell activity during HIV infection: a decrease in NK activity is observed at the clonal level and is not restored after in vitro long-term culture of NK cells.

A Critical Role for Type I IFN–dependent NK Cell Activation in Innate Immune Elimination of Adenoviral Vectors In Vivo

Effector cells mediating natural killer (NK) cell activity and antibody‐dependent cellular cytotoxicity (ADCC) have been receiving considerable interest recently as they may represent an additional cellular defense mechanism against maglinancy. Spleen cells from normal young C3H (MTV+) female mice were found to have relatively high natural killer (NK) activities toward RL 1 and YAC‐1 tumor cell lines and high antibody‐dependent cell‐mediated cytotoxicity (ADCC) activities toward chicken RBC and SB tumor targets. Serum inhibition of NK and ADCC activities was either not demonstrable or only seen at low levels in the young mice. Following injection of a transplantable mammary adenocarcinoma that grew progressively in these mice, both NK and ADCC activities decreased and serummediated inhibition of both NK and ADCC increased as the tumors grew. NK and ADCC activities declined with age in C3H (MTV+) mice, and the serum inhibition of NK and ADCC activities of young mice increased with age. Aging mice, however, which had developed spontaneous mammary adenocarcinoma, although having increased levels of inhibitor against both NK and ADCC activities and decreased NK and ADCC activities of their spleen cells as compared to young normal mice, did not have different levels from those of aging mice which had not developed the mammary tumor. Correlation was poor between the amount of serum inhibitor(s) on NK and ADCC activities. NK versus RL 1 and ADCC for chicken RBC were not correlated, but NK versus YAC‐1 and ADCC for the tumor cell line SB appeared to be correlated. Decreasing NK and ADCC and increasing serum inhibitory activity toward these functions appeared to correlate with tumor progression. Sera from transplantable tumor‐bearing young C3H mice contained increased amounts of circulating immune complexes, but the amount of circulating immune complexes was only weakly correlated with the serum inhibition of NK activity of spleen cells from young animals by sera from tumor‐bearing young mice. These findings indicate that a serum blocking factor(s) that can inhibit natural cell‐mediated cytotoxicity or antibody‐mediated cytotoxicity in vitro must be considered in analyzing host resistance to tumor growth.

Mechanism of NK activation by OK-432 ( Streptococcus pyogenes) : I. Spontaneous release of NKCF and augmentation of NKCF production following stimulation with NK target cells

Differential staphylococcal protein a-induced enhancement of natural killer cell activity of lymphocytes from HIV-seropositive individuals

Natural (or “spontaneous”) cytotoxicity against the K-562 myeloid cell line and antibody-dependent cellular cytotoxicity (ADCC) against sensitized Chang liver cells were monitored simultaneously in a 4 hr 51Cr release assay, using the same preparations of normal human peripheral blood lymphocytes as effector cells. Both types of cytotoxic activity were expressed by lymphocytes bearing receptors for sheep erythrocytes and for the Fc portion of IgG. Up to 80% of the total natural killer (NK) cell and K cell (ADCC) activities were recovered in E-rosette pellets separated under optimal conditions. Since the ability to form rosettes with sheep erythrocytes is primarily a marker for human T cells, it seems likely that both NK and K cells are in the T cell lineage. Further, both NK and K cell cytolytic activities were severely depressed after brief exposure of lymphocyte suspensions or rosette pellets to ammonium chloride solutions. Both activities returned to normal values after 24-hr incubation at 24°C. Such sensitivity of human lymphocytes to ammonium chloride solutions may explain why other investigators have failed to observe NK or ADCC activity by E-RFC. When lymphocytes bearing Fc receptors were adsorbed onto plastic surfaces coated with a monolayer of immobilized antigen-antibody complexes made up of TNP and rabbit anti-TNP serum, both NK and ADCC activities were simultaneously depleted. Such depletion was completely blocked when Staphylococcal protein A, which binds selectively to the Fc portion of IgG, was incubated on the antigen-antibody monolayers before adding the lymphocytes. This confirmed that both NK and ADCC activities were mediated by effector lymphocytes bearing Fc receptors. When protein A at concentrations ranging from 0.01 to 100 µg/ml was added to the 51Cr release assays, ADCC was significantly inhibited, presumably due to binding of protein A to available Fc sites of the sensitizing antibody. However, protein A had no effect on NK reactivity at any of the concentrations tested. When lymphocytes were treated for 30 min at 37°C with purified trypsin or α-chymotrypsin at concentrations from 0.01 to 10 mg/ml, NK activity was significantly inhibited whereas ADCC was unaffected. Other enzymes depressed (pronase, subtilisin) had no effect except at high concentrations (lipase), or stimulated (neuraminidase) both NK and K cell activity similarly, and therefore failed to distinguish between the effector cell types. Thus, our data indicate that effector cells in NK and ADCC are in overlapping, if not identical, subpopulations of Fc receptor-bearing lymphocytes of probable T cell lineage. The cytolytic mechanisms in NK and ADCC appear to be distinct, since we have been unable to find evidence for an “arming” antibody involved in this NK system.

Changes in Both CD4+CD25+Foxp3+ Regulatory T Cells and CD3-CD56+ NK Cells in Patients with Chronic Myeloid Leukemia Treated with a Tyrosine Kinase Inhibitor

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease with few therapeutic options. However, the immune system, including natural killer (NK) cells, is linked to ALS progression and may constitute a viable therapeutic ALS target. Tofacitinib is an FDA-approved immunomodulating small molecule which suppresses immune cell function by blocking proinflammatory cytokine signaling. This includes the cytokine IL-15 which is the primary cytokine associated with NK cell function and proliferation. However, the impact of tofacitinib on NK activation and cytotoxicity has not been thoroughly investigated, particularly in ALS. We therefore tested the ability of tofacitinib to suppress cytotoxicity and cytokine production in an NK cell line and in primary NK cells derived from control and ALS participants. We also investigated whether tofacitinib protected ALS neurons from NK cell cytotoxicity. Finally, we conducted a comprehensive pharmacokinetic study of tofacitinib in mice and tested the feasibility of administration formulated in chow. Success was assessed through the impact of tofacitinib on peripheral NK cell levels in mice. We found tofacitinib suppressed IL-15-induced activation as measured by STAT1 phosphorylation, cytotoxicity, pro-inflammatory gene expression, and pro-inflammatory cytokine secretion in both an NK cell line and primary NK cells. Furthermore, tofacitinib protected ALS neurons from NK cell-mediated cytotoxicity. In mice, we found tofacitinib bioavailability was 37% in both male and female mice; using these data we formulated mouse containing low and high doses of tofacitinib and found that the drug suppressed peripheral NK cell levels in a dose-dependent manner. These results demonstrate that tofacitinib can suppress NK cell function and may be a viable therapeutic strategy for ALS.

Natural killer (NK) cells can lyse certain tumor cells as well as virally infected cells without prior sensitization.Animal studies have shown that general anesthesia (GA) alone or GA followed by surgery decreases both basal NK cytotoxicity and enhancement of NK activity by interferon (IFN). The purpose of this study was to determine whether similar inhibition of NK activity by anesthesia and surgery occurs in humans. Venous blood was drawn 1 h before and 20-24 h after surgery under isoflurane/N2 O anesthesia. Peripheral blood mononuclear cells (PBMC) were assayed for basal and IFN-alpha-stimulated NK cytotoxicity using a chromium release assay with K562 cells as targets. Flow cytometry was used to enumerate NK, T-helper, and T-cytotoxic/suppressor cell populations in each sample. Basal NK activity was significantly depressed after GA and GA and surgery. Although the postoperative IFN treatment increased NK activity to the preoperative basal level, the level achieved was significantly lower than the level observed after IFN stimulation of PBMC as evaluated preoperatively. This decreased activity does not seem to be the result of a decrease in the percentage of circulating NK cells. The decrease in NK activity after anesthesia and surgery may lead to increased susceptibility to infection and/or tumor dissemination and thus needs to be explored. Implications: Natural killer cells can kill cancer cells and virally infected cells. This study shows that surgery with general anesthesia leads to decreased natural killer cell activity as assessed in the laboratory. This decreased natural killer cell activity may lead to infection or tumor dissemination. NK activity can be restored to presurgery levels by treating isolated NK cells with interferon-alpha. (Anesth Analg 1997;85:918-23)

Natural killer (NK) cell responses are crucial for anti-viral and anti-tumor immunity. However, the immune responses of NK cells are impaired in chronically HCV-infected patients. While direct-acting antivirals (DAAs) treatment allows a complete viral clearance in HCV patients, the recovery of NK cell immunity is only fragmentary explored and underlying molecular mechanisms are fundamentally uncharacterized. The first part of this thesis aims to characterize the molecular phenotype of NK cells in chronic HCV patients before and after DAA treatment by using quantitative proteomics. In total, more than 4867 proteins were identified and quantified by mass spectrometry, covering both the surface markers and intracellular components. Principal component analysis and heatmap of correlation factors revealed that NK cell proteomes of the same patients one year after treatment did normalize their inter-individual variance to the level of healthy individuals but, notably, were found still different from the healthy controls. The dysregulated proteins before and after treatment complement our knowledge of impaired NK cell responses in chronically HCV infected patients and indicated both trained and impaired NK cell functions in cured patients. Among protein regulations that indicated trained NK cell immunity, the adhesion and degranulation-promoting adaptor protein (ADAP) was selected for functional studies. Thus, the second part of this thesis investigated the importance of ADAP in human NK cells. In vitro experiments showed that the Src kinases-associated phosphoprotein 1 and 2 are interaction partners of ADAP. Moreover, ADAP regulates cytokine production and degranulation upon the stimulation of K562 cells, as well as migration and adhesion upon stimulation of CXCR4. Interestingly, ADAP is also required for controlling the number of serially killed target cells for each NK cell and the ratio of cytotoxic NK cells. To sum up, these data show that ADAP promotes serial killing through regulating adhesion and migration in human NK cells, confirming that the up-regulation of ADAP is a part of trained immunity in cured HCV patients.

Because alterations in natural killer (NK) activity in the perinatal period may be important in the maintenance of a healthy pregnancy, we examined the mechanisms by which these alterations are mediated in neonates and in pregnant and postpartum women. NK activity, as measured in a 4-hr 51Cr-release assay and compared with adult controls, is significantly diminished in all three trimesters of pregnancy and in immediately postpartum women. In postpartum women, NK activity appears to be higher than in pregnant women, although this does not reach statistical significance. Pregnant and postpartum women have normal numbers of large granular lymphocytes and normal target cell binding in an agarose single cell assay but decreased lysis of the bound target cells. NK activity of mononuclear cells from postpartum women, in addition, demonstrate a shift in distribution to higher levels of resistance to gamma-irradiation. Further, sera from postpartum women cause a similar shift to increased radioresistance in mononuclear cells from adult controls. Because radioresistance is a property of interleukin 2-stimulated NK, the shift to radioresistance may represent lymphokine-mediated stimulation occurring during parturition. In contrast, cord blood cells have a more profound decrease in NK activity as determined by 51Cr-release assay and decreases in both binding and lysis of bound target cells in the single cell assay. The resistance of NK activity in cord cells to gamma-irradiation is also increased, as seen in postpartum women. Cord blood serum, however, did not alter radioresistance or inhibit NK activity. The results suggest that the observed diminished NK activity in pregnant women and neonates arise by different mechanisms: an absence of mature NK cells in the neonate and an alteration of the NK cell in pregnancy leading to decreased killing.

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system that are capable of killing virally infected and/or cancerous cells. Nearly 20 years ago, NK cell-mediated immunotherapy emerged as a safe and effective treatment approach for patients with advanced-stage leukaemia. Subsequently, the field of NK cell-based cancer therapy has grown exponentially and currently constitutes a major area of immunotherapy innovation. In general, the development of NK cell-directed therapies has two main focal points: optimizing the source of therapeutic NK cells for adoptive transfer and enhancing NK cell cytotoxicity and persistence in vivo. A wide variety of sources of therapeutic NK cells are currently being tested clinically, including haploidentical NK cells, umbilical cord blood NK cells, stem cell-derived NK cells, NK cell lines, adaptive NK cells, cytokine-induced memory-like NK cells and chimeric antigen receptor NK cells. A plethora of methods to augment the cytotoxicity and longevity of NK cells are also under clinical investigation, including cytokine-based agents, NK cell-engager molecules and immune-checkpoint inhibitors. In this Review, we highlight the variety of ways in which diverse NK cell products and their auxiliary therapeutics are being leveraged to target human cancers. We also identify future avenues for NK cell therapy research.

Severe acute respiratory syndrome coronavirus 2 induced coronavirus disease 2019 (COVID-19) has posed a great challenge to public health worldwide and also increased susceptibility to colorectal cancer (CRC). Natural killer (NK) cells serve as the first line of defense in the host's innate immune system, performing natural killing functions and mediating cytotoxicity against tumors and viruses. Therefore, a better understanding of NK cell cytotoxicity may facilitate the development of treatment strategies for CRC-associated with COVID-19. To investigate the cytotoxic killing function of peripheral NK cells in patients with CRC and severe COVID-19 (CRC+ patients). The percentages of circulating NK and NKT cells in CRC+ and age-matched patients with CRC were analyzed using flow cytometry. NK cell cytotoxic activity (NKCA) and corresponding NK cytotoxic factor (NKCF) activity in peripheral blood mononuclear cells were evaluated using a Real-Time Cell Analyzer. The numbers and percentage of peripheral NK and NKT cells in patients with CRC+ were lower than those in patients with CRC. Additionally, compared to patients with CRC, those with CRC+ had lower levels of NKCA and NKCF activity in lysed K562 cells. Positive correlations were observed between NKCA and NK cell numbers, NKCA and NK cell percentages, NKCF activity, and NK cell percentages in patients with CRC+. Furthermore, a negative correlation was observed between NKCA and the severity of COVID-19 in patients with CRC. The area under the receiver operating characteristic curve for NKCA was greater than those for the other indices. CRC+ is associated with lower levels of peripheral NK cells and impaired natural cytotoxicity, contributing to the immunopathogenesis of severe COVID-19 rather than immune control.

NKG2C Improves Diagnostic Specificity of Natural Killer (NK) Cell Receptor Restriction by Identifying Nonneoplastic Adaptive NK Cell Clones.

Recent reports revealed that dendritic cell (DC)-natural killer (NK) cell interaction plays an important role in tumor immunity, but few DC vaccine studies have attempted to evaluate the non-specific, yet potentially clinically relevant, NK response to immunization. In this study, we first analyzed in vitro activation of NK cells by DCs similar to those used in clinical trials. Subsequently, NK cell responses were analyzed in a phase I clinical trial of a vaccine consisting of autologous DCs loaded with a fowlpox vector encoding CEA. The data were compared with the clinical outcome of the patients. DC enhances NK activity in vitro, partly by sustaining NK cell survival and by enhancing the expression of NK-activating receptors, including NKp46 and NKG2D. Among nine patients in our clinical trial, NK cytolytic activity increased in four (range 2.5-5 times greater lytic activity) including three who had increased NK cell frequency, was stable in two and decreased in three. NKp46 and NKG2D expression showed a good correlation with the patients' NK activity. When patients were grouped by clinical activity (stable disease/no evidence of disease (stable/NE, n=5) vs progressive disease (N=4) at 3 months), the majority in the stable/NE group had increases in NK activity (P=0.016). Anti-CEA T cell response was enhanced in all the nine patients analyzed, but was not significantly different between the two groups (P=0.14). Thus, NK responses following DC vaccination may correlate more closely with clinical outcome than do T cell responses. Monitoring of NK response during vaccine studies should be routinely performed.

The natural killer (NK) and antibody-dependent cellular cytotoxic (ADCC) activities were determined in peripheral blood lymphocytes of patients with gynecologic malignancy. The effects of a preparation of Streptococcus pyogenes (OK-432) and of interferon were determined. Patients with advanced cancer exhibited lower natural cytotoxicity than normal women. NK and ADCC activities were decreased following surgery and NK activity was decreased following chemotherapy. Radiation reduced the percentage of Leu-7-positive cells in the circulation of the patients. OK-432 augmented the NK activity of both healthy women and patients with early malignancy. Interferon was less effective than OK-432 in augmenting NK activity.