Natural Killer Cell Functional Activity Research Articles

Natural Killer Cell Dysfunction in Premenopausal BRCA1 Mutation Carriers: A Potential Mechanism for Ovarian Carcinogenesis.

Tissue-specificity for fimbrial fallopian tube ovarian carcinogenesis remains largely unknown in BRCA1 mutation carriers. We aimed to assess the cell autonomous and cell-nonautonomous implications of a germline BRCA1 mutation in the context of cancer immunosurveillance of CD3- CD56+ natural killer (NK) cells. Premenopausal BRCA1 mutation carriers versus age-matched non-carriers were compared. Daily urinary 5β-pregnanediol levels were used to determine progesterone metabolomics across an ovarian cycle. Using peripherally acquired NK cells the cell-mediated cytotoxicity of tumor targets (OVCAR-3, K-562) was determined using live cellular impedance (xCELLigence®) and multicolor flow cytometry. Hypoxia-inducible factor 1-alpha (HIF-1α) immunohistochemistry of cancer-free fallopian tube specimens allowed a comparison of proximal versus distal portions. Utilizing these findings the role of environmental factors relevant to the fimbrial fallopian tube (progesterone, hypoxia) on NK cell functional activity were studied in an ovarian phase-specific manner. BRCA1 mutation carriers demonstrate a differential progesterone metabolome with a phase-specific reduction of peripheral NK cell functional activity. Progesterone exposure further impairs NK cell-mediated cytotoxicity in a dose-dependent manner, which is reversed with the addition of mifepristone (1.25 µM). The fimbrial fallopian tube demonstrated significantly higher HIF-1α staining, particularly in BRCA1 mutation carriers, reflecting a site-specific 'hypoxic niche'. Exposure to hypoxic conditions (1% O2) can further impair tumor cytotoxicity in high-risk carriers. Phase-specific differential NK cell activity in BRCA1 mutation carriers, either systemically or locally, may favor site-specific pre-invasive carcinogenesis. These cumulative effects across a reproductive lifecycle in high-risk carriers can have a detrimental effect further supporting epidemiological evidence for ovulation inhibition.

Dendritic Cell-Based Vaccines Recruit Neutrophils to the Local Draining Lymph Nodes to Prime Natural Killer Cell Responses

Dendritic cell (DC)-based cancer vaccines are a form of immunotherapy that activates the innate and adaptive immune systems to combat cancers. Neutrophils contribute to cancer biology and have the potential to be exploited by immunotherapeutic platforms to enhance anti-tumor immune responses. We previously showed that DC vaccines elicit the expansion of mouse interferon (IFN)γ-producing mature natural killer (NK) cells to elevate anti-tumor responses. Here, we demonstrate the rapid recruitment of neutrophils to the draining lymph nodes of DC-vaccinated mice. This was accompanied by an increase in the total number of NK cells producing IFNγ and expressing CD107a, a marker of degranulation that demonstrates NK cell functional activity. Furthermore, the depletion of neutrophils in DC-immunized mice resulted in decreased numbers of NK cells in draining lymph nodes compared to the controls. Interestingly, the increased number of IFNγ- and CD107a-expressing NK cells in DC-immunized mice was not detected in mice depleted of neutrophils. Further investigations showed that DC vaccines induced IFNγ- and TNFα-producing CD8+ T cells that also expressed CD107a, but depletion of neutrophils did not have any impact on the CD8+ T cell population. Our findings suggest that neutrophil-mediated anti-tumor immunity induced by a DC vaccine platform could be targeted to provide innovative strategies to enhance its clinical efficacy.

SIRT2 Promotes Murine Melanoma Progression Through Natural Killer Cell Inhibition

SIRT2, an NAD+-dependent histone deacetylase, has been shown to play a pivotal role in various physiological processes, however, its role in cancer is currently controversial. In recent years, SIRT2 has been described as both a tumor suppressor and oncogene with divergent expression and function in various malignancies. With established roles in inflammation and glucose and iron metabolism, systemic and microenvironment SIRT2, rather than just intrinsic tumor cell expression, might have implications in tumor progression. Moreover, recent interest in the tumor immune microenvironment has further characterized immune cells involved in tumor suppression, including natural killer (NK) cells. Here, we sought to investigate the biological role of systemic SIRT2 in melanoma tumor progression and to evaluate its interaction with key tumor suppressive immune cells.Murine models were employed to investigate the effect of systemic SIRT2 on melanoma tumor progression. Tumor progression was measured in terms of weight and volume for 20 days following subcutaneous inoculation of Sirt2-KI and WT mice with B16-F10 melanoma cells. Differences in tumor-infiltrating immune cell composition were determined by flow cytometry. Granzyme B and Ki-67 immunohistochemical staining was used to measure NK cell functional activity and proliferation in in vivo melanoma samples. NK cell function was further evaluated using in vitro and ex vivo mouse models as well as an in vitro human model. Antibody-mediated NK cell depletion established NK cells as an integral player in SIRT2-mediated tumor promotion. Finally, SIRT2 was pharmacologically inhibited by SirReal2, and the effect on melanoma progression and NK cell tumor infiltration was established.Increased systemic expression of SIRT2 promoted melanoma progression in mice, resulting in larger and heavier tumors. Mechanistically, systemic SIRT2 overexpression reduced the number of tumor-infiltrating NK cells and suppressed NK cell activation and proliferation within the tumor microenvironment. Furthermore, despite the effect of increased tumor growth rate and tumor volume in wild-type littermate mice, NK cell depletion did not affect that in SIRT2-overexpressing mice. Lastly, pharmacological inhibition of SIRT2 increased NK cell tumor infiltration and suppressed melanoma tumor growth.Systemic SIRT2 and NK cell activity exhibit a dynamic functional interaction which promotes melanoma tumor progression. Given the recent renewed interest in NK-cell-mediated immunotherapy response, SIRT2 could represent a new opportunity to mediate immunotherapy response and resistance.

P–386 Uterine Natural Killer Cell function in Recurrent Miscarriage and Implantation Failure: A Systematic Review

Abstract Study question Does uterine natural killer cell functional activity differ in women with recurrent miscarriage (RM) or implantation failure (RIF) compared to fertile controls? Summary answer There is insufficient data to conclusively determine differences in uterine natural killer (NK) cell activity between women with RM/RIF and controls. What is known already Uterine NK cell (uNK) function is central to maintaining healthy pregnancy by promoting placentation and spiral artery vascular remodelling. The range of uNK activity is diverse and includes cytokine secretion, such as IFN-γ, or angiogenic factors which promote vascular remodelling. Despite an abundance of studies investigating peripheral blood NK cell cytotoxicity, there is limited evidence of uNK cytotoxicity. uNK-trophoblast interactions are facilitated by uNK receptors such as CD94, LILRB1 and KIRs. It is possible that dysfunction of these diverse uNK activities plays a more important role in early pregnancy failure than uNK levels. Study design, size, duration We conducted a systematic review of prospective case-control studies investigating uterine natural killer cell activity in patients with RM or RIF versus controls. The aim was to determine whether there was a distinct variation in uNK activity between RM/RIF and controls. We stratified uNK activity into four broad categories: i) regulation and receptors; ii) cytotoxicity; iii) expression of cytokines; iv) effect on uterine vasculature. Participants/materials, setting, methods The electronic database search included MEDLINE, EMBASE, Web of Science and bibliographies from included articles from inception to December 2020 using a combination of MESH and keywords. Search, screen, and data extraction were performed by two reviewers independently. Quality assessment was conducted with ROBINS-I. Out of 4636 studies screened, 30 studies (1696 women) were analysed for uNK activity. Main results and the role of chance Different methods were used to measure uNK activity including immunohistochemistry, flow cytometry, ELISA, PCR and Western blot. Samples were obtained from endometrium during mid-luteal phase or decidua following surgery. 14 studies reported on uNK phenotypes associated with regulation and receptors. RM/RIF patients, compared to controls, demonstrated a reduced expression of KIR2DL4, KIR2DL3/L2/S2 and inhibitory receptors (NKG2A); whereas there was a higher expression of the activating receptor (NKp46) and CD161. One study reported correlation between FoxP3+ T-cells and CD56+ NK cells but another reported no similar correlation with CD57+/CD56+ ratio. Two studies investigating dNK cytotoxicity, using chromium release or lactate dehydrogenase release assays, concluded higher dNK cytotoxicity in RM patients. Eight studies reported on cytokine expression. Interestingly, two studies found lower expression of IFN-g, but four studies reported otherwise in RM patients. Two studies found a higher ratio of dNK producing IFN-γ/TNF-α to those producing IL–4. The rest of the studies reported lower expression of IL–1RA, IL–10, TNF-α, Lnc–49A and higher expression of granzyme B, perforin and PRF–1. Finally, six studies reported on effect of uNK on vasculature. Among the findings were negative correlation between CD16+ uNK and endometrial IL–6 and VEGF, as well as higher angiogenin, VEGF and bFGF expression. Limitations, reasons for caution Functional activity investigated amongst studies varied significantly; this heterogeneity precluded meta-analysis of the data. Heterogeneity also prohibited definitive conclusions on uNK function and pregnancy outcome. Studies presented data in a combination of qualitative and quantitative analysis and variation was seen in the criteria for inclusion of RM, RIF and controls. Wider implications of the findings: uNK levels alone may be insufficient to guide management of early pregnancy failure. Clarification of underlying functional activity may guide therapeutic intervention and help develop new treatments. This review highlights the need for a greater understanding of the role of uNK activity in healthy pregnancy and early pregnancy failure. Trial registration number N/A

Hepatitis C virus-induced NK cell activation causes metzincin-mediated CD16 cleavage and impaired antibody-dependent cytotoxicity

The Fc receptor family for immunoglobulin (Ig)G type III (FcγRIII, CD16) is an activating receptor on natural killer (NK) cells and an essential mediator of antibody-dependent cellular cytotoxicity (ADCC). There is only limited information on its role during chronic hepatitis C virus (HCV) infection. We studied CD16 expression in relation to NK cell functional activity in HCV-infected patients and sought mechanistic insights into virus-induced modulation. NK cell CD16 expression and activation status were evaluated ex vivo by flow cytometry in HCV-infected patients and healthy controls (HC) as well as in vitro after co-culture with HCV-infected HuH7.5 cells. Rituximab-mediated ADCC was assessed in HC and HCV-infected patients using Daudi cells as a target. The role of metzincins in CD16 down-modulation was assessed using specific inhibitory molecules and by evaluating intracellular mRNA levels. HCV-infected patients exhibited increased frequencies of ex vivo activated NK cells and a concomitantly decreased NK CD16 expression, which resulted in impaired ADCC activity. Moreover, exposure of NK cells to culture-derived HCV recapitulated the ex vivo findings of decreased CD16 expression and increased NK cell activation. Importantly, blockade of metzincin-mediated shedding activity, including selective a disintegrin and metalloproteinase 17 (ADAM-17) inhibition, restored NK CD16 expression. Successful treatment with direct-acting antivirals partially improved NK ADCC function despite delayed CD16 reconstitution. Chronic HCV infection induces NK cell activation resulting in ADAM-17-dependent CD16 shedding and consequent impaired ADCC function. Altered ADCC may contribute to failure to eradicate HCV-infected hepatocytes. We show here that hepatitis C virus (HCV) activates natural killer (NK) lymphocytes which, as a consequence, loose their Fc receptor for IgG (CD16), an essential molecule for antibody binding. We show that this occurs through the action of enzymes named metzincins, resulting in altered NK-mediated antibody-dependent killing (ADCC) of target cells. This mechanism may contribute to HCV persistence and may represent a general phenomenon whereby some viruses can escape host's immune responses.

Combination of Anti-PD-L1 Antibody with IMiD® Immunomodulatory Drugs, HDAC Inhibitor ACY-1215, Bortezomib, or Toll-like Receptor 9 Agonist Enhances Anti-Tumor Immunity and Cytotoxicity in Multiple Myeloma

Introduction Dysfunctional T cells and Natural Killer (NK) cells in MM, together with functionally defective plasmacytoid dendritic cells (pDCs), contribute to the immune suppression in MM (Chauhan et al, Cancer Cell 2009, 16:309-323; Ray et al, Leukemia 2014, 28: 1716-1724). The mechanism and the role of immunoregulatory molecules mediating pDC-T cell and pDC-NK cell interactions in MM are now defined. Programmed cell death protein 1 (PD-1) is highly expressed on MM patient T cells and NK cells; and both pDCs and MM cells express PD-1 ligand PD-L1 (B7-H1). PD-L1 interaction with PD-1 results in bidirectional inhibitory responses in T cells. Our study showed that pDCs confer T cell and NK cell immune suppression in the MM BM milieu by engaging immune checkpoints via PD-L1/PD-1 signaling axis (Ray et al, Leukemia 2015, 29:1441-1444). Importantly, blockade of PD-L1-PD-1 using anti-PD-L1 Ab generates MM-specific CD8+ CTL activity, as well as enhances NK-cell-mediated MM cell cytolytic activity. Anti-MM therapies may modulate MM-host immune responses, which raises the possibility that efficacy of anti-PD-Ll Ab can be improved by combining these therapies with immune-stimulating agents. Here we examined the impact of combining immune checkpoint blockade with lenalidomide, pomalidomide, bortezomib, HDAC inhibitor ACY-1215, or Toll-Like Receptor 9 agonists on anti-tumor immunity and cytotoxicity in MM.Methods For combination studies, we utilized low concentrations of various drugs (pomalidomide, lenalidomide, ACY-1215, or bortezomib) that do not significantly decrease viability of MM cells. As in our prior studies, anti-PD-L1 Ab and TLR9 agonist are not cytotoxic against MM cells. T cell proliferation assay: MM patient pDCs were co-cultured with autologous T cells (pDC:T ratio; 1:10) in the presence of anti-PD-L1 Ab (5 μg/ml) alone, drug alone, or anti-PD-L1 Ab plus drug for 5-6 days, and proliferation was quantified with CellTrace Violet Cell proliferation Kit using FACS. CTL activity assays: MM patient CD8+ T cells were cultured with autologous pDCs (1:10 pDC/T ratio) with anti-PD-L1 Ab, drug alone, or anti-PD-L1 plus drug for 5 days; cells were washed to remove drug, and GFP+MM.1S cells (20:1 E/T ratio) were added for another 2-3 days, followed by quantification of viable GFP+MM.1S cells using FACS. NK-cell mediated cytotoxic activity was assessed using flow-based CFSE-stained K562 lysis assays, as well as degranulation assay quantifying cell surface CD107a. All statistical parameters were calculated using GraphPad Prism 6. Anti-PD-L1 Ab was purchased from eBiosciences, USA; and ACY-1215, bortezomib, lenalidomide, and pomalidomide were purchased from Selleck chemicals, USA.Results Combination of anti-PD-L1 Ab (5 μg/ml) with lenalidomide (50-100 nM) or pomalidomide (100 nM) triggered a more robust MM-specific CD8+ CTL activity than anti-PD-L1 Ab alone (1.5-2 and 2-3 fold increase in CTL activity for lenalidomide and pomalidomide combinations, repectively). Anti-PD-L1 Ab combination with lenalidomide or pomalidomide also significantly increased NK-cell-mediated MM cell cytotoxicity (p < 0.05). We next determined whether anti-PD-L1 Ab can be combined with histone deacetylase inhibitors ACY-1215 (250 nM) or Panobinostat (2 nM). Combination of anti-PD-L1 Ab with ACY-1215 or panobinostat enhanced MM-specific CD8+ CTL activity versus anti-PD-L1 Ab alone (1.5 and 2 fold increase in CTL activity for panobinostat and ACY-1215 combinations, respectively). Assessment of surface CD107a as a marker of NK cell functional activity showed that anti-PD-L1 Ab plus ACY-1215 markedly increased CD107a expression (>10 fold) versus anti-PD-L1 Ab alone. Our prior studies showed that TLR9 agonists can restore pDCs ability to trigger T cell proliferation. We found that a combination of anti-PD-L1 Ab and TLR9 agonists (1 μM) enhances MM-specific pDC-induced CTL activities (2-3 fold increase in CTL activity in combination regimen versus anti-PD-L1 Ab alone). Finally, a combination of bortezomib (2 nM) with anti-PD-L1 Ab increased the MM-specific CTL activity (1.5-2 fold increase).Conclusions Our study provides the basis for combining novel immunotherapies targeting PD-1/PD-L1 pathway with current anti-MM agents or pDC-activating TLR agonists, to both restore immune function and enhance cytotoxicity in MM.Corresponding Author: Dharminder Chauhan, PhD DisclosuresChauhan:Stemline Therapeutics: Consultancy.

Glucocorticoid influence on IFNγ production and the epigenetic landscape

Emotional distress is a common response to psychological stress. Such distress activates the hypothalamic pituitary adrenal axis resulting in excessive glucocorticoid production, which profoundly impacts immune function. We have modeled this impact by in vitro treatment of human peripheral blood mononuclear cells with glucocorticoids in order to assess the relationships among immune function and epigenetic post translational modifications. This was accomplished by evaluation of the nuclear intensity and localization of the repressive epigenetic mark H3K27me3 and the cytoplasmic production of the cytokine interferon gamma (IFN γ ). Glucocorticoid treatment significantly increased the detectable levels of H3K27me3 and significantly decreased the levels of IFN γ in both the CD56 bright NK and CD56 dim NK cell populations. There were also significant increases in the number of cells with an increased localization of H3K27me3 to the nuclear periphery (consistent with heterochromatic DNA) which coincided with decreases in IFN γ production. Both H3K27me3 MFI and the localization of H3K27me3 toward the nuclear periphery are negatively correlated with cytoplasmic IFN γ production in glucocorticoid treated samples. These results demonstrate glucocorticoids reduce IFN γ production by natural killer cells via the heterochromatic re-localization of H3K27me3 to the nuclear periphery. Measurement of H3K27me3 to this heterochromatic region of the nucleus may provide an index of reduced natural killer cell functional activity.

Natural Killer Cell Functional Activity After 4-1BB Costimulation.

Reports show enhancement of CD8 T cells' activity through CD137 (4-1BB) signal; however, not all data proved similar effect in natural killer (NK) cells. Here, the impact of 4-1BB signal on NK cells' function was assessed during short term cultures. To that end, cytokine-activated NK cells were cocultured with adenovirally transduced MCF-7 stimulator cells expressing 4-1BB ligand. Cellular cytotoxicity, cytokine production, and expression of cytotoxicity related genes were assessed after overnight cultures. Sharp decrease of CD56+ and CD56bright NK cells was demonstrated. 4-1BB neither enhanced cellular degranulation nor improved IFN-γ production although it promoted granzyme B, perforin, and FasL gene expression. 4-1BB signal stimulated higher proportions of CD56bright population to degranulate and express CD107a; however, it could not recover killing activity against K562 targets. Our data could not show major promotion in activity of all NK subpopulations. Due to great heterogeneity of NK cells, more investigation is needed to draw a comprehensive conclusion.

Inhibition of human natural killer cell functional activity by human aspartyl β-hydroxylase.

Natural killer (NK) cells are a key component of the innate immune system and play pivotal roles as inflammatory regulators and in tumor surveillance. Human aspartyl β-hydroxylase (HAAH) is a plasma membrane and endoplasmic reticulum protein with hydroxylation activity, which is over-expressed in many malignant neoplasms and can be detected from the sera of tumor patients. HAAH is involved in regulating tumor cell infiltration and metastasis. Escaping from immune surveillance may help tumor cell infiltration and metastasis. However, the effects of HAAH on tumor immune surveillance have not yet been investigated carefully. The present study investigated the potential use of HAAH as an immune regulator of human NK cells. We assessed the effects of recombinant HAAH (r-HAAH) on primary human NK cell morphology, viability, cytotoxicity, apoptosis, receptors expression and cytokine/cytolytic proteins production. Our results demonstrated that r-HAAH negatively affects NK cell activity in a time and dose-dependent manner. It noticeably reduces the viability of the NK cells by increasing apoptosis and necrosis via caspase signaling pathways. Moreover, r-HAAH reduces the NK cell cytotoxicity by inhibiting surface expression of NKG2D, NKp44 and IFN-γ secretion. These findings suggest that one of the ways by which HAAH actively promotes tumor formation and proliferation is by inhibiting NK cell-surveillance activity.

Natural Killer Cells from Patients with Chronic Rhinosinusitis Have Impaired Effector Functions

Natural killer (NK) cells are multicompetent lymphocytes of the innate immune system that play a central role in host defense and immune regulation. Although increasing evidence suggests that innate immunity plays a key role in the pathogenesis of chronic rhinosinusitis (CRS), the role of NK cells in CRS has been poorly studied. This study aimed to characterize the peripheral blood NK cells from patients with CRS, and to compare the functions of these cells with those from non-CRS controls. The correlation between NK cell functional activity and prognosis was also assessed. Eighteen CRS patients and 19 healthy non-CRS controls were included. The patients with CRS were classified into two subgroups, namely a treatment-responsive group and recalcitrant group. NK cell degranulation was determined by measuring the cell surface expression of CD107a against 721.221 and K562 cells. Intracytoplasmic cytokine production was determined by flow cytometry. Compared to the controls, the NK cells of CRS group had an impaired ability to degranulate and to produce cytokines such as IFN-γ and TNF-α. The recalcitrant subgroup showed the most severe defects in NK cell effector functions. Moreover, the decreased NK cell functions in patients with CRS were associated with poor prognostic factors such as concomitant asthma and peripheral blood eosinophilia. NK cells, which were originally named for their ability to mediate spontaneous cytotoxicity towards diseased cells including infected cells, may play an important role in regulating the inflammatory process in CRS pathogenesis.

Phase 1 Clinical Trial of Trametes versicolor in Women with Breast Cancer

Introduction. Orally administered preparations from the Trametes versicolor (Tv) mushroom have been hypothesized to improve immune response in women with breast cancer after standard chemotherapy and radiotherapy. Methods. A phase I, two-center, dose escalation study was done to determine the maximum tolerated dose of a Tv preparation when taken daily in divided doses for 6 weeks after recent completion of radiotherapy. Eleven participants were recruited and nine women completed the study. Each cohort was comprised of three participants given one of three doses of Tv (3, 6, or 9 grams). Immune data was collected pre- and postradiation, at 3 on-treatment time points and after a 3-week washout. Results. Nine adverse events were reported (7 mild, 1 moderate, and 1 severe), suggesting that Tv was well tolerated. Immunological results indicated trends in (1) increased lymphocyte counts at 6 and 9 grams/day; (2) increased natural killer cell functional activity at 6 grams/day; (3) dose-related increases in CD8+ T cells and CD19+ B cells , but not CD4+ T cells or CD16+56+ NK cells. Conclusion. These findings show that up to 9 grams/day of a Tv preparation is safe and tolerable in women with breast cancer in the postprimary treatment setting. This Tv preparation may improve immune status in immunocompromised breast cancer patients following standard primary oncologic treatment.

Natural Killer Cells Derived From Human Pluripotent Stem Cells Provide a Novel Method to Treat HIV-1 Infection.

Abstract 280Natural killer (NK) cells are known to be key components of the innate immune system with the ability to kill diverse tumor cells and virally infected cells. Our group has previously demonstrated derivation of CD45+CD56+ natural killer (NK) cells from human embryonic stem cells (hESCs)-derived hematopoietic CD34+CD45+ progenitor cells. These hESC-derived NK cells demonstrate potent killing of various tumor cells both in vitro and in vivo. More recently, we have also successfully generated NK cells from similar CD34+CD45+ hematopoietic progenitor cells derived from human induced pluripotent stem cells (iPSCs). Again, we find that these iPSC-derived NK cells also have effective anti-tumor activity in vitro. Notably, both the hESC and iPSC-derived NK cells are uniformly CD94+CD117−, corresponding to a more mature and cytotoxic NK cell population. This is in contrast to NK cells derived from umbilical cord blood (UCB) progenitor cells that produce a mixture of CD94+CD117− and CD94−CD117+ NK cells that are more heterogeneous in their cytotoxic activity. Previous studies of NK cells isolated from peripheral blood indicate they have activity against HIV-1-infected cells. Therefore, we hypothesized that both hESC and iPSC-derived NK cells would be able to kill HIV-1-infected targets. We have applied multiple complementary systems to test this hypothesis using both HIV-1-infected cell lines and HIV-1-infected primary T cells. First, we used a chronically infected cell line (H9/HTLV IIIB) to demonstrate specific cytolytic activity of hESC-derived NK cells. Here, we found CD107a expression (a marker of NK cell functional activity) was significantly upregulated on hESC-derived effectors stimulated by the HIV-1-infected targets compared to uninfected targets (13.7% vs. 4.3%). Next, we utilized primary human CD4 T cells infected with primary patient isolate HIV96-480 as targets to demonstrate the same effect to specifically activate both hESC and iPSC-derived NK cells. In both of these studies, control NK cell populations derived from human umbilical cord blood progenitor cells were significantly less active against HIV than the hESC and iPSC-derived NK cells. In addition to the cytolytic function against HIV-1-infected targets, we demonstrate hESC and iPSC-derived NK cells also suppress HIV-1 replication by producing CC-chemokines to competitively inhibit CCR5 co-receptor binding. CCL4 (MIP1b), a CCR5 ligand, is greatly induced in both hESC and iPSC-derived NK cells after incubation with HIV-1-infected targets compared to uninfected targets: 37.2% (HIV-infected) vs. 24.8% (uninfected) for hESC-NK cells and 32.5% (HIV-infected) vs. 21.6% (uninfected) for iPSC-NK cells. Lastly, we have also tested suppression of acute HIV-1 infection by hESC-derived NK cells in vitro. Here, the T-cell line CEM-GFP was infected with HIV-1 NL4-3 and cocultured with hESC-derived NK cells for two weeks. HIV-1-infected targets detected by flow cytometry for GFP expression were strongly decreased in comparison to controls in absence of NK cells (% of GFP+ cells: 0.74 vs. 26.4). In these analyses, expression of CD107a and CCL4 on the effectors again correlates with the inhibition of HIV-1 replication. Currently, as hESC and iPSC-derived NK cells express the Fc receptor CD16, we are using anti-envelope protein antibodies against HIV-1 infected primary human CD4 T cells to determine if antibody-dependent cellular cytotoxicity provides another mechanism of lytic activity for these novel NK cells. Collectively, our results demonstrate that NK cells derived from hESCs and iPSCs provide an effective novel cellular immunotherapy for HIV-1 infection. Disclosures:No relevant conflicts of interest to declare.

CD107a as a functional marker for the identification of natural killer cell activity

Natural killer (NK) cells are a subset of lymphocytes that play a central role in the innate immune response to tumors and infections. An important limitation in the field of NK research is attributable to the deficit of assays available for the detection of the functional activity of NK cells. Recently, lysosomal-associated membrane protein-1 (LAMP-1 or CD107a) has been described as a marker of CD8 + T-cell degranulation following stimulation. Here we describe CD107a as a marker of NK cell functional activity using multi-parameter flow cytometry. CD107a is significantly upregulated on the surface of NK cells following stimulation with MHC devoid targets. Additionally, CD107a expression correlates with both cytokine secretion and NK cell-mediated lysis of target cells. However, as well as being coordinately expressed on nearly all cytokine secreting cells, CD107a was also expressed on a large subset of NK cells that did not secrete cytokine following stimulation. These data suggest that employing CD107a as a marker of NK cell functional activity may allow for the identification of a large fraction of activated NK cells that may degranulate in the absence of cytokine secretion. Cumulatively, the data presented here demonstrate that CD107a is a sensitive marker of NK cell activity.

Clinical relapses of multiple sclerosis are associated with ‘novel’ valleys in natural killer cell functional activity

Natural killer (NK) cells are implicated in the pathogenesis of multiple sclerosis (MS). Nine relapsing–remitting MS (RRMS) patients along with age, sex, and NK responder status matched controls were studied serially. Although the average NK cell functional activity (FA) was not significantly different between both groups, four clinical relapses in RRMS patients were associated with the development of ‘novel’ valleys in FA. These valleys are of greater depth and duration than cyclical valleys observed in both RRMS and controls, precede the onset of clinical attacks, and are observed in RRMS but not controls. In both RRMS and controls, cyclical peaks and valleys in FA are determined by the number of CD33 +, CD3 −CD56 +, and to a lessor extent CD3 +CD56 + cells capable of binding targets and inducing cell-mediated cytotoxicity (CMC). In contrast, ‘novel’ valleys in FA result from a reduction in the ability of CD3 −CD56 + bound to targets to induce CMC. The results suggest that RRMS patients are at greater risk for clinical relapses during ‘novel’ valleys in FA. Furthermore, these valleys are the result of cells with a NK cell phenotype being unable to deliver a ‘lethal’ hit to targets.

Individual differences in personality, immunology and mood in patients undergoing self‐hypnosis training for the successful treatment of a chronic viral illness, HSV‐2

AbstractIndividual differences in personality, in particular cognitive activation, withdrawal and hypnotic susceptibility were examined in a study of self‐hypnosis training in patients with chronic and severe herpes simplex virus (HSV‐2), genital herpes, along with depression, anxiety and activation ratings. Immune parameters were assayed before and after the six‐week training. Self‐hypnosis almost halved recurrence rate, benefiting 65% of the patients. Furthermore clinical responders demonstrated an increase in natural killer cell (NKC) cytotoxicity of herpes infected cells as well as up‐regulation of nonspecific immune parameters. In replication of a student exam stress study (Gruzelier, Smith, Nagy and Henderson, 2001a) cognitive activation was positively associated with immune function; with improvement in herpes recurrence, improvements in specific NKC cytotoxicity, baseline functional NKC activity and energy ratings, and was unrelated to frequency of practice. No other measure of personality or affect was associated with immune system up‐regulation and improvements in health. Depression and, to a lesser extent, anxiety improved independently of herpes outcome. Depression and/or anxiety at baseline were positively associated with withdrawal, particularly the affective aspects, and neuroticism, which were associated with immune down‐regulation seen in NKC counts and NKC functional activity at baseline. Hypnotizability was associated positively with aspects of immune system up‐regulation and negatively with clinical depression. Immune system up‐regulation in clinical responders correlated positively with frequency of self‐hypnosis practice. This is possibly the first evidence for the efficacy of self‐hypnosis training for a chronic medical illness. Evidence (1) of the benefits to health and (2) of the viral specificity of the immune changes, here in the form of increased cytotoxicity of NKC for cells infected with the herpes virus, gives credence to the value of a psychological intervention for immunity; evidence for which is scarce in the field of psychoneuroimmunology. The predictive ability of cognitive activation for response to hypnotherapy is in keeping with the preferential effects on both immune parameters and health of hypnosis instructions which require the generation of dynamic imagery in contrast to instructions of passive relaxation imagery (Gruzelier, Levy, Williams and Henderson, 2001b). Copyright © 2002 British Society of Experimental and Clinical Hypnosis

A role for natural killer cells in the immunopathogenesis of multiple sclerosis

Seventeen relapsing–remitting (R/R) multiple sclerosis (MS) patients and age/sex matched controls were studied every 6 weeks for 2 years. Disease activity, determined both clinically and by serial MRI, was correlated with natural killer (NK) cell functional activity (FA) and phenotype. Mean NK cell FA is significantly lower in MS patients, compared to controls ( P<0.001), while variability around the means is significantly greater ( P<0.01). The spectrum of mean NK cell FA, observed in the patient cohort, along with cyclical nature of the FA and phenotype over time, observed in both patients and controls, may begin to explain the discrepant results reported in previous studies. In R/R MS, there is a significant correlation between reductions (valleys) in NK cell FA and the development of active lesions on MRI, new ( P<0.001) or enlarging ( P=0.05). More importantly, a significant number of active lesions, new ( P=0.01) and enlarging ( P=0.02), are preceded by a reduction in NK cell FA. The correlation between the onset of clinical attacks and valleys of NK cell FA is also significant ( P=0.002). When taken together, the results suggest that reductions (valleys) in NK cell FA represent periods of susceptibility for the development of active lesions on MRI and clinical attacks. A significant positive correlation is also identified between mean NK cell FA for each R/R MS patient and total number of active MRI lesions developed by that patient over the 2 years ( P=0.001). The results would suggest that R/R MS patients with a higher mean NK cell FA are at greater risk for the development of active lesions. These results support the proposal that NK cells may play a role in the immunopathogenesis of R/R MS.

Immunocompetence of Tissue Infiltrating Lymphocytes in Bladder Tumors

Tissue infiltrating lymphocytes (TIL) in bladder tumors have been assumed to be an expression of local host resistance against the tumor. We investigated the functional activity of TIL compared to peripheral blood lymphocytes (PEL). Isolation of TIL was performed using the enzyme cocktail treatment with Ficoll-Hypaque discontinuous gradient centrifugation.Analysis of lymphocyte subsets by flow cytometry demonstrated Leu 4, 43.6% (T cells); Leu 10, 10.5% (B cells) and Leu 7, 13.1% (natural killer (NK) cells) in TIL. The cytotoxic activity of TIL and PEL was tested in a four hour 51Cr-release assay. Myeloid K562 cells (NK sensitive), HT 1197 (bladder tumor) and fresh bladder tumors were used as target cells. The spontaneous NK cell activity of PBL was 23.7%, whereas that of TIL was only 3.5%. However, in vitro culture with IL2 induced a significant augmentation of NK activity in TIL as well as in PBL. On the other hand, the spontaneous lymphokine activated killer cell (LAK) activity of PBL and TIL was very low. Uncultured PBL and TIL exhibited the highest levels of lysis against fresh bladder tumors. Unlike PBL, IL2-induced cytotoxicity of TIL against autologous bladder tumors was higher than that against allogenic bladder tumors.Immunomodulators OK432 and 112 were injected intratumorally during endoscopy. Analysis of the lymphocyte subsets in TIL showed an increase of T and NK cells following immunomodulator injection. Endoscopic injection of immunomodulators into bladder tumors augmented NK cell functional activity in TIL as well as PBL.These findings suggest that local immunosurveillance is directed against bladder tumors. Further studies are required to understand more fully the local and systemic host immune responses in cancer.

Study on lymphocyte subsets and NK cell function in patients with dilated cardiomyopathy.

An acquired defect or damage of a subpopulation of suppressor T lymphocytes is reported in connection with autoimmune diseases. In the present study, the role of immunity was examined in 7 patients with dilated cardiomyopathy (DCM). The frequency of lymphocyte subsets using monoclonal antibodies and natural killer (NK) cell activity was evaluated to determine whether DCM patients had lymphocyte abnormalities that would support the hypothesis that the pathological mechanism of DCM is an immune disturbance. The peripheral lymphocyte counts were significantly lower in patients with DCM and higher in patients with ischemic heart disease (IHD) than in normal controls (NC) (p less than 0.01). The percentage of T cells, B cells, OKT4 and OKT8 positive cells was not statistically different among the three groups studied here, whereas the percentage of T gamma cells was significantly reduced in DCM patients (p less than 0.05). NK cell functional activity as tested in DCM and IHD patients was frequently deficient (22.1 +/- 19.3% in DCM, 13.8 +/- 3.0% in IHD, 37.4 +/- 12.7% in NC). Our results suggest that an imbalance in cellular immune reactions partly explain the pathogenesis of DCM.

Quantitation of lymphocyte subsets by immunofluorescence flow cytometry in idiopathic dilated cardiomyopathy

Idiopathic dilated cardiomyopathy (IDC) is a disease in which immune aberration has been postulated but not confirmed. The frequency of lymphocyte subsets was evaluated in 22 patients with IDC and in 22 blood bank control subjects, using monoclonal antibodies to cell surface markers to allow cell sorting by immunofluorescence flow cytometry. Eighteen patients with heart failure from other causes were also studied. Functional correlations were also made for the natural killer cell subset. Total T-cell frequency, determined with antihuman T (Hybritech), was similar in IDC and control groups: mean 73 ± 12% in IDC patients and 70 ± 9 % in control subjects. B-cell frequency, determined with antihuman la (Hybritech), was also similar: 36 ± 11 % in IDC patients and 31 ± 10% in control subjects. Helper T-cell frequency, identified by OKT4 (Ortho), averaged 47 ± 11 % in IDC and 44 ± 8 % in control subjects (difference not significant). Suppressor/cytotoxic T-cell frequency, established by OKT8, was the same: 28 ± 8 % in IDC and 30 ± 7 % in control subjects, although relative deficiency in suppressor functional activity has been reported in IDC. Helper to suppressor (OKT4/8) ratios, aberrant in many autoimmune diseases, did not differ significantly (IDC 1.9 ± 0.8, control 1.5 ± 0.6). Lymphocyte subsets and OKT4/8 ratio were also similar between IDC and heart failure patients. Natural killer cell frequencies, estimated using 2 antibodies (antihuman Leu-7 and Leu-11, Becton-Dickinson) were the same (9.3 ± 4.9 % in IDC patients, 9.0 ± 4.5 % in control subjects, Leu-7). In contrast, natural killer cell functional activity was decreased in the IDC group; median lymphocyte to target cell (K562 line) ratio resulting in 50% killing (L/T50) was 21 in IDC vs 10.5 in control (p <0.02). Macrophage (OKM1) frequency also did not differ. It is concluded that, despite marked cardiac dysfunction in IDC, relative percentages of lymphocyte subsets, identified by cell surface markers, are not consistently changed. In contrast, functional deficiencies (as for natural killer cells) may be present, suggesting abnormalities in activation that may respond to pharmacologic maneuvers.

Immunology of tumor infiltrating lymphocytes.

Frequently peripheral blood lymphocytes (PBL) do not reflect the tumor host relationship and cell mediated immunity in the PBL does not often correlate with prognosis. The tumor infiltrating lymphocytes (TIL) interact most closely with the tumor cells and are likely to more accurately reflect tumor host interactions. These studies indicate that TIL from pulmonary tumors are similar to PBL so far as their cell surface markers are concerned. The percentage of T-cells, B-cells, helper cells, suppressor cells, and NK cells are similar in the two compartments. However, the TIL are markedly suppressed in their functional capacity as measured by their proliferative and cytotoxic activity. In addition, natural killer (NK) cell activity is markedly diminished in TIL as opposed to the PBL. In addition, the direct injection of BCG into these tumors reverses this phenomenon by significantly increasing T-cell and NK cell functional activity. Thus, the microenvironment of the tumor profoundly affects the immunologic relationship between the tumor and the host.