Innate immune response of the human host to exposure with herpes simplex virus type 1: in vitro control of the virus infection by enhanced natural killer activity via interleukin-15 induction

Infections with herpes simplex virus type 1 (HSV-1) in humans and in animal models are accompanied by enhanced natural killer (NK) activity. In vitro, HSV-1 also enhances the NK activity of human peripheral blood mononuclear cells (PBMC). The molecular basis of this enhanced NK activity, however, is not well characterized. We investigated the role of human interleukin-15 (IL-15) in this phenomenon and report here that HSV-1-mediated enhanced NK activity was abrogated by neutralizing antibodies for IL-15 but not for other cytokines (i.e., IL-2, IL-12, gamma interferon [IFN-gamma], tumor necrosis factor alpha, or IFN-alpha). Anti-CD122 antibodies which block signaling through IL-2 receptor beta chain, and therefore neutralize the effects of IL-15 (and IL-2), also abrogated this enhancement. Furthermore, HSV-1 increased the levels of IL-15 mRNA and the production of IL-15 in HSV-1-infected PBMC cultures. The neutralization of IL-15 in cocultures of PBMC with HSV-1-infected cells significantly increased HSV-1 production. These results strongly suggest a role for IL-15 in the HSV-1-mediated in vitro enhancement of NK activity and in the PBMC-mediated suppression of HSV-1 replication.     

Prostaglandin E2 inhibits human T-cell proliferation after crosslinking of the CD3-Ti complex by directly affecting T cells at an early step of the activation process

We have studied the effects of prostaglandin E2 (PGE2) on in vitro human T-cell activation induced by crosslinking of the CD3-Ti complex with the monoclonal anti-CD3 antibodies OKT3 and UCHT-1. PGE2 (greater than or equal to 3 X 10(-9) M) when added simultaneously with anti-CD3 to cultures of peripheral blood mononuclear cells (PBMC), significantly suppressed, in a dose-dependent way, T-cell proliferation (P less than 0.002). However, when T cells were first preactivated with OKT3 for 3 days, subsequent proliferation driven by recombinant interleukin 2 (IL-2) was not inhibited by addition of PGE2. This indicates that PGE2 affects the activation step resulting from crosslinking of CD3-Ti, but not the IL-2-driven proliferative phase. Other manifestations of T-cell activation were therefore examined. Both IL-2 production and the expression of receptors for IL-2 (as detected with the anti-Tac monoclonal antibody) were inhibited by PGE2. The addition of purified interleukin 1 (IL-1) or recombinant IL-2 to the cultures did not reverse the inhibiting effect of PGE2 on IL-2-receptor expression. PGE2, added at the time of culture initiation, also inhibited T-cell proliferation in cultures which were supplemented with exogenous IL-1 or IL-2. Proof for a direct effect of PGE2 on T cells was obtained in experiments in which monocyte-depleted T cells were stimulated, in the presence of IL-1, with solid-phase-bound anti-CD3 antibody. Proliferation of T cells in this system is accessory cell independent and still was strongly inhibited by PGE2. Finally, preincubation of PBMC with PGE2 (3 X 10(-6) M) for 48 hr did not result in the generation of suppressor cells for anti-CD3-induced T-cell proliferation or for IL-2 production. Our results demonstrate that PGE2 has a direct inhibitory effect on an early step of T-cell activation, resulting in decreased IL-2 production, decreased IL-2-receptor expression, decreased responsiveness to exogenous IL-2, and decreased proliferation. However, PGE2 does not affect IL-2-driven proliferation of activated T cells. The inhibitory effect on T-cell activation is not mediated through suppressor T cells, nor through inhibition of accessory cell function.     

Induction of cytotoxicity against autologous tumour cells by interleukin-12: evidence for intrinsic anti-tumor immune capacity in curatively resected gastrointestinal tumour patients

The aim of this study was to investigate the cell-mediated immune response in 14 patients undergoing curative resection for a gastrointestinal tumor by the induction of peripheral blood mononuclear cell (PBMC)-mediated immune activity against autologous tumour cells. PBMC were stimulated by interleukin-12 (IL-12; 100 IU/ml) and IL-2 (1,000 IU/ml) without contact with tumour cells for 36 h. Specific cytotoxic activity against autologous tumour cells (auTu), natural killer (NK)-sensitive cells (K562) and allogeneic tumour cells (RF48/HT29) was determined by fluorescence cytotoxicity assay. Additionally, inhibition experiments using the mononuclear antibodies (mAb) FMC16 and W6/32 against major histocompatibility complex I (MHC I) on autologous tumour cells were performed in order to determine the involvement of specific T lymphocytes. The cytotoxic activity of unstimulated PBMC did not differ between the three target cells. IL-12 caused a 3.2-fold increase in activity against auTu ( P=0.002). In contrast, after stimulation with IL-2, only a slight increase in activity was observed. After IL-12 stimulation, cytotoxic activity against auTu was 2.5- to 2.7-fold higher than the corresponding activity against K562/allogeneic tumour cells ( P=0.002/ P=0.006). After blocking of the MHC I complex on auTu by FMC 16 or W6/32 mAb, a 62.9%/74.4% reduction in the specific cytotoxicity of IL-12-stimulated PBMC was found. In summary, IL-12 induced an effective immune response against auTu, which was partly mediated by specific cytotoxic T lymphocytes (CTL). It was considered that de novo generation of this activity during 36 h incubation without antigen contact was hardly possible, but that the observed induction of effective anti-tumor cytotoxicity was rather based on the re-activation of a pre-existing immune potential from the tumour-host interaction. These findings indicate the existence of an autologous anti-tumor immune response following curative resection in patients undergoing surgery for solid tumours, which might influence the development of tumour recurrence from disseminated tumour cells. Making use of this capacity could constitute an attractive immunotherapeutical approach for curatively operated tumour patients.     

The role of natural killer (NK) cells and NK cell receptor polymorphisms in the assessment of HIV-1 neutralization

The importance of innate immune cells in HIV-1 pathogenesis and protection has been highlighted by the role of natural killer (NK) cells in the containment of viral replication. Use of peripheral blood mononuclear cells (PBMC) in immunologic studies provides both HIV-1 target cells (ie. CD4+ T cells), as well as anti-HIV-1 effector cells, such as NK cells. In this study, NK and other immune cell populations were analyzed in HIV-negative donor PBMC for an impact on the anti-HIV activity of polyclonal and monoclonal antibodies. NK cell percentages were significantly higher in donor PBMC that supported lower levels of viral replication. While the percentage of NK cells was not directly associated with neutralization titers, NK cell-depletion significantly diminished the antiviral antibody activity by up to three logs, and polymorphisms in NK killer immunoglobulin receptor (KIR) and FcγRIIIa alleles appear to be associated with this affect. These findings demonstrate that NK cells and NK cell receptor polymorphisms may influence assessment of traditional HIV-1 neutralization in a platform where antibody is continuously present. This format appears to simultaneously assess conventional entry inhibition (neutralization) and non-neutralizing antibody-dependent HIV inhibition, which may provide the opportunity to delineate the dominant antibody function(s) in polyclonal vaccine responses.     

Lactobacillus strains induce TRAIL production and facilitate natural killer activity against cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an endogenous cytokine that induces apoptosis in malignant tumor cells. Here, we show for the first time that lactobacilli induce TRAIL production in human peripheral blood mononuclear cells (PBMC). Treatment with lactobacilli induced TRAIL on the cell surface of PBMC and in culture medium. The TRAIL production induced by lactobacilli partially depends on IFN-α and IFN-γ. Lactobacilli treatment facilitated NK activity of PBMC against prostate cancer cells. Moreover, TRAIL neutralization antibody efficiently prevented the NK activity. Our results indicate that lactobacilli facilitate NK activity through TRAIL production, and raise the possibility of a new TRAIL-based strategy against malignant tumors.     

Regulation of human natural cytotoxicity by IgG. III. Interaction between negative regulation by monomeric IgG and positive regulation by interferons of different types

Our prior reported results have demonstrated the dose-dependent inhibition of human natural killer (NK) cell activity upon treatment of peripheral blood mononuclear cells (PBMC) with monomeric IgG (mIgG) prior to the cytotoxic assay. In the present study, the combined effects on NK activity of human interferon (IFN) of each of the three types and mIgG, respectively, were determined. NK cells incubated with IFN alpha or IFN beta had augmented cytotoxicity against K562 target cells but remained responsive to negative regulation by mIgG. PBMC treated with human recombinant IFN gamma had unchanged cytotoxic activity but became partially resistant to suppression by mIgG. This ability of IFN gamma to interfere with the negative regulation of NK activity by cytophilic mIgG was seen when the cytokine was preincubated with effector cells prior to, simultaneously with, or after their exposure to inhibitor protein. These data provide some clues regarding the possible biological significance of the mIgG-induced down-regulation of NK cells which, when required for host protection, might be appreciably reversed or blocked by IFN gamma produced by NK cells or T cells in response to various agents.     

Cell-mediated immune response in human hepatic echinococcosis. Analysis of antigen-induced lymphoproliferation and NK activity

In vitro cellular immune responsiveness was studied in 25 patients undergoing surgery for hepatic hydatid disease and in 22 matched healthy controls. Proliferation of peripheral blood mononuclear cells (PBMC) induced by phytohaemagglutinin (PHA) was not statistically different in the two groups, while proliferation induced by antigenic preparations obtained from the human commensal microorganism Candida albicans was depressed in patients as compared to healthy subjects. Confirming previous data, antigen specific proliferative response to hydatid cyst fluids was greatly enhanced in patients as compared to controls (P less than 0.01). On the other hand, natural killer (NK) activity was significantly reduced (P less than 0.005). Both impairment of NK activity and lymphoproliferation induced by commensal microorganisms suggest that patients following the parasitic infection present a condition of relevant hyporesponsiveness in cell-mediated defence.     

A macrophage derived blastogenic factor -MBF- induces cytokines and cytotoxic effectors in human peripheral blood mononuclear cells.

A soluble macrophage-derived blastogenic factor, previously reported as MBF, is secreted from macrophages activated with galactose oxidase. It was previously shown that MBF is able to induce IFN-gamma production and proliferation of T lymphocytes. In this study we found that MBF is able to induce in human peripheral blood mononuclear cells (PBMC) production of interleukin 1 (IL-1) beta, interleukin 2 (IL-2) and tumor necrosis factor (TNF) alpha and generation of MHC-unrestricted cytotoxic activity. The induction of killer cells is likely to rely on IFN-gamma production in that in PBMC treated with a monoclonal antibody (Mab) against IFN-gamma, the MBF induced cytotoxic activity was drastically reduced. A comparison of MBF induced cytotoxic effectors with those induced by IL-2 showed that both cytotoxic effectors pertain to NK lineage, in that they were CD3- and CD16+. On the contrary, the precursors of MBF and IL-2 induced killer cells were different; MBF cytotoxic precursor cells were highly sensitive to L-Leucine methyl ester (Leu-OME), a drug able to eliminate monocytes and NK cells, whereas IL-2 cytotoxic precursors were unaffected by this drug.     

Induction of human NK cell-mediated cytotoxicity by CD40 triggering on antigen presenting cells

Engagement of CD40 on antigen presenting cells (APC) is central to the initiation of cell-mediated immune response. Here, we investigated the ability of CD40 ligation on APC to induce NK cell-mediated cytotoxicity in the human system and the mechanism(s) underlying this process. We showed that APC (consisting in adherent peripheral blood mononuclear cells) (PBMC), pre-stimulated with anti-CD40 monoclonal antibodies and co-cultured with autologous non-adherent PBMC for 5-9 days, induced CD3-/CD56+ NK cell-mediated cytotoxicity as well as CD3+/CD56+ T cell-mediated unrestricted cytotoxic activity. The generation of NK cell-mediated cytotoxicity was independent on cell-to-cell contact between CD40-triggered APC and NK cells. Moreover, we found that IL-12 did not play a role in NK cells induction by anti-CD40 priming, while IL-2 and IL-15 did play a role. Our results provide an insight into the mechanism by which NK cells are activated in peripheral blood and useful informations for therapeutic application of anti-CD40 antibodies.     

Purine nucleoside modulation of functions of human lymphocytes.

The accumulation of endogenous substrates in patients with adenosine deaminase deficiency or purine nucleoside phosphorylase deficiency is believed to be responsible for the immunodeficiency observed in these patients. To identify the lymphocyte populations that are most susceptible to these substrates, we investigated the effect of their nucleoside analogs on a number of T and B cell functions of human lymphocytes. We found that tubercidin (Tub), 2-chloro 2'deoxyadenosine (2CldA), 2-fluoro adenine arabinoside-5'phosphate (FaraAMP), and 9-beta-D-arabinosyl guanine (AraGua) inhibited the proliferative responses of human peripheral blood mononuclear cells (PBMC) to polyclonal activators (PHA, OKT3 mab) or to allogeneic PBMC in mixed lymphocyte cultures (MLC). Addition of recombinant IL-2 from the beginning of the culture did not alter the inhibition by Tub of the proliferative responses of PBMC. These purine nucleoside analogs also inhibited the proliferative responses of purified human peripheral blood CD4+ and CD8+ T cells to PHA and of purified B cells to SAC. The concentrations of these nucleosides required to achieve a given degree of inhibition of proliferative responses of T lymphocyte subpopulations or B cells was similar, suggesting that these analogs do not exhibit any selectivity for these purified lymphocyte populations. Tub and FaraAMP, respectively, inhibited and enhanced, at the effector phase, both NK cytotoxicity and specific T cell-mediated cytotoxicity. In contrast to these findings, LAK cytotoxicity at the effector phase was not significantly inhibited by Tub, and was not enhanced by FaraAMP. Both analogs inhibited rIL-2-induced proliferative responses of PBMC, but did not affect the generation of LAK cytotoxicity (induction phase) against the K562 targets when added at the beginning of the culture. This suggests that DNA synthesis is not required for LAK cell induction. Both Tub and FaraAMP inhibited immunoglobulin production (IgG and IgM) by PBMC in the PWM-induced system. These results demonstrate that purine nucleoside analogs significantly inhibited a number of functions of human lymphocytes. Although selectivity for T lymphocyte subpopulations and B cells was not observed, a differential effect of Tub and FaraAMP on LAK cytotoxicity versus NK cytotoxicity and specific T cell cytotoxicity was found.     

Correlation of human CD56+ cell cytotoxicity and IFN-gamma production

The use of an IFN-gamma ELISPOT assay to evaluate cellular immune responses has gained increasing popularity, especially as a surrogate measure for cytotoxic T lymphocyte (CTL) responses. We have compared the IFN-gamma ELISPOT assay and the traditional(51)Cr release assay for detection of human natural killer (NK) cell activity. The cell populations used for evaluation of these assays included freshly isolated and IL-2-activated peripheral blood mononuclear cells (PBMC). CD56-positive cells were demonstrated to be the primary source of the IFN-gamma signal when PBMC were evaluated with NK-sensitive targets in the IFN-gamma ELISPOT assay. IFN-gamma ELISPOT and(51)Cr release assays showed excellent correlation suggesting that NK activity can be reliably evaluated with methods other than the traditional(51)Cr release assays.     

Biological activity of 26,26,26,27,27,27-hexafluorinated analogs of vitamin D3 in inhibiting interleukin-2 production by peripheral blood mononuclear cells stimulated by phytohemagglutinin

Vitamin D compounds suppress the production of interleukin-2 (IL-2) by peripheral blood mononuclear cells (PBMCs) stimulated with phytohemagglutinin in a dose-dependent manner. We used this suppression to test 26,26,26,27,27,27-hexafluorinated analogs of vitamin D3 for their immunosuppressive activity in PBMCs. 26,26,26,27,27,27-Hexafluoro-1,25-dihydroxyvitamin D3 and 26,26,26,27,27,27-hexafluoro-1,24-dihydroxyvitamin D3 were approximately 10 times more potent than 1,25-dihydroxyvitamin D3 in suppressing IL-2 production. 26,26,26,27,27,27-Hexafluoro-1-hydroxyvitamin D3 was 20 to 30 times less potent than 1,25-dihydroxyvitamin D3 in causing this effect. The relative biopotency of each vitamin D3 analog toward PBMC proliferation was roughly similar to that toward IL-2 production by PBMCs. Suppression of PBMC proliferation by vitamin D3 analogs seemed to be a secondary effect of their inhibition of IL-2 production.     

Effects of resveratrol on human immune cell function.

Resveratrol (3,5,4'-trihydroxystilbene), a polyphenol found in grapes and grape products such as red wine, has been reported to exhibit a wide range of biological and pharmacological activities both in vitro and in vivo. Because many of the biological activities of resveratrol, like the inhibition of cyclooxygenase, induction of CD95 signaling-dependent apoptosis, effects on cell division cycle and modulation of NF-kB activation, suggest a possible effect on the immune system, we evaluated the in vitro effects of resveratrol in three immune response models: i) development of cytokine-producing CD4+ and CD8+ T cells induced by stimulation of peripheral blood mononuclear cells (PBMC) with anti-CD3/anti-CD28; ii) specific antigen-induced generation of cytotoxic T lymphocytes; iii) natural killer (NK) activity of PBMC. The results showed that in vitro exposure to resveratrol produces a biphasic effect on the anti-CD3/anti-CD28-induced development of both IFN-gamma- IL2- and IL4-producing CD8+ and CD4+ T cells, with stimulation at low resveratrol concentrations and suppression at high concentrations. Similarly, the compound was found to induce a significant enhancement at low concentrations and suppression at high concentrations of both CTL and NK cell cytotoxic activity. On the whole, the results of the study indicate that resveratrol modulates several human immune cell functions and suggest that this activity may be related to its effects on cytokine production by both CD4+ and CD8+ T cells.     

Cellular immunosuppression in children with acute lymphoblastic leukemia: Effect of consolidation chemotherapy

Summary The present study was designed to evaluate the chemotherapy-induced cellular immunosuppression in 20 children with acute lymphoblastic leukemia (ALL) in remission and receiving maintenance chemotherapy. Peripheral blood was serially obtained from leukemic children during vincristine/cyclophosphamide/6-mercaptopurine/prednisone combined consolidation chemotherapy. The mean absolute number of peripheral blood lymphocytes as well as the mean absolute numbers of lymphocyte subsets (T cells, T cell subsets, B cells, and natural killer cells) from leukemic children before consolidation chemotherapy were all significantly lower than in control subjects; however, the percentages of lymphocyte subsets were similar in both groups. After consolidation chemotherapy, the percentages of CD4⁺ T lymphocytes and natural killer (NK) cells were significantly decreased and the percentages of monocytes and CD8⁺ T lymphocytes were significantly increased. Phytohemagglutinin- and 12-O-tetradecanoylphorbol-13-acetate-induced production of interleukin-2 (IL-2) and NK-cell-mediated cytotoxic activity by peripheral blood mononuclear cells (PBMC) were also substantially decreased in the post-therapy groups. NK activity correlated with the percentage of NK cells in PBMC. In contrast, OK432-induced production of tumor necrosis factor (TNF) and killer activity against NK-resistant target cells were significantly increased after therapy as compared with the pre-therapy and control groups. TNF production correlated with the percentage of monocytes in PBMC. These results demonstrate that substantial quantitative and qualitative chemotherapy-induced abnormalities of the cellular immune system are present in the majority of patients treated with ALL. It is also suggested that the increased TNF production by monocytes and the appearance of potent killing activity against NK-resistant targets might compensate for the defects of IL-2 production and NK activity during intensive consolidation chemotherapy.This work was supported by a grant-in-aid for cancer research from the Ministry of Health and Welfare, Japan, and a grant-in-aid from the Association for the Support of Children with Cancer     

Inert particles inhibit natural killer cell function in vitro

Aqueous suspensions of inert particles were found to inhibit the baseline and interferon-enhanced natural killer (NK) cell activity of peripheral blood mononuclear cells (PBMC) and large granular lymphocytes (LGLs). This inhibition was induced with latex, silica, and Sephadex particles. The suppression of NK activity was not related to effector cell death as determined by trypan blue exclusion. The inhibition of NK cell function was more pronounced with prolonged incubation and could be partially reversed with monocyte depletion or the addition of indomethacin, a prostaglandin synthesis inhibitor, but not with the addition of the lipoxygenase inhibitors nordihydroguaiaretic acid and BW755C. Similarly, particle exposure inhibited the NK cell function of monocyte-depleted large granular lymphocytes with and without the add-back of glass adherent cells, implying that monocyte-independent NK suppressive mechanisms were also present. These data demonstrate that inert particles are immunosuppressive in vitro and can inhibit baseline and interferon-stimulated NK cell function of LGLs and PBMC through monocyte-dependent and independent pathways.     

Specific inhibition effects of N-pentafluorobenzyl-1-deoxynojirimycin on human CD4+ T cells

We first synthesized N-pentafluorobenzyl-1-deoxynojirimycin (5F-DNM), one new derivative of 1-deoxynojirimycin (DNM). Effects on human peripheral blood mononuclear cells (PMBC) and secretion of cytokines from human PBMC by 5F-DNM were investigated. It was first found that 5F-DNM remarkably inhibited the secretion of interleukin-4 (IL-4) and had a specific inhibition on the expression of CD4 molecules. 5F-DNM, much less toxic than cyclosporin A, might be used as a new candidate of immunosuppressant for specifically treating Th2-mediated immune diseases.     

Natural killer cell activity correlates with the amount of beta 2-microglobulin on human peripheral blood lymphocytes

Two cytotoxic assays, lectin-dependent cytotoxicity and natural killer (NK) cytotoxicity, were used to assess the competence of cord blood and neonatal peripheral blood mononuclear cell (PBMC) and T-cell cytotoxic reactions. The effect of exogenous interferon was also studied. Results were compared with cytotoxic capabilities of adult cells and cells from patients with primary immunodeficiency syndromes. Lectin-dependent cytotoxicity (LDCC), a property of both T and non-T cells, was assessed by lysis of chromium-labeled EL4 tumor target cells in the presence or absence of exogenous fibroblast interferon (IFN-β). Natural killer cytotoxicity was assessed by lysis of two different chromium-labeled tumor target cells, Molt 4f and K562 in the presence or absence of IFN-β. Lectin-dependent cytotoxicity (LDCC) of PBMC of cord blood (32 ± 4% SEM) and adult cells (36 ± 2% SEM) were equivalent but neonatal cells had slightly decreased LDCC (22 ± 3% lysis). T-depleted cells from cord or neonatal blood had increased LDCC but T-enriched (>95% sheep erythrocyte rosette-forming cells) from both cord (22 ± 3%) and neonatal blood (18 ± 5%) had significantly reduced LDCC compared to 55 ± 2% for adult T cells. This deficiency corrects with age and is near normal after age 2. Preincubation with IFN-β did not enhance LDCC of newborn or adult cells. The LDCC of some cord T cells was markedly reduced and was in the same low range as patients with severe combined immunodeficiency. Natural killer (NK) cytotoxicity of PBMC from cord and adult cells was equivalent at three effector:target ratios against the Molt 4f target but against the K562 target, cord PBMC had significantly less NK activity (22 ± 11 SD) compared to adult NK activity (50.5 ± 22.2 SD) at a 50:1 effector:target ratio. Similar differences were noted at 25:1 and 10:1 target:effector ratios. NK cytotoxicity against Molt 4f targets of adult cells was significantly enhanced by preincubation with IFN-β but NK of cord cells was only variably enhanced. By contrast, IFN-β enhanced NK against K562 targets of both adult and cord cells, adult greater (67.7 ± 20) than cord cells (37.8 ± 2.0). These T-cell effector deficiencies are in marked contrast to the vigorous proliferative responses of newborn T cells, and parallel deficiencies of certain neonatal lymphokines. These defects may explain the newborns' enhanced susceptibility to intracellular viruses and to congenital viral infections.     

Difference in response of NK cell activity in newborns and adult to IL-2, IL-12 and IL-15

The killing activity of cord blood mononuclear cells (cMNC) against cytomegalovirus (CMV)-uninfected and -infected fibroblasts was comparable to that of adult peripheral blood mononuclear cells (aPBMC). The killing activity of cMNC against K562 cells was significantly lower compared with that of aPBMC. Treatment of cMNC and aPBMC with interleukin-2 (IL-2), IL-12 or IL-15 significantly enhanced killing activity against K562 cells and CMV-uninfected and -infected cells. By comparison of cMNC with aPBMC, killing activity against the K562 cells of cMNC was augmented to the level of aPBMC when cultured with IL-2, IL-12 or IL-15. The killing activity of cMNC against CMV-uninfected and -infected fibroblasts did not increase to the level of adult PBMC by treatment with IL-2, IL-12 or IL-15. These data suggest that cord blood contains a functionally different NK cell subpopulation than that among adult NK cells.