Effect Of Peripheral Blood Mononuclear Cells Research Articles

In vitro and Molecular Docking Analysis of Quercetin as an Anti-inflammatory and Antioxidant.

Quercetin (3,3',4',5,7-pentahydroxyflavone) is a dietary flavonoid with good antioxidant and anti-inflammatory properties. The present study aims to determine these effects in peripheral blood mononuclear cells (PBMCs) evoked by lipopolysaccharides (LPS). The mRNA expression and protein secretion of inflammatory mediators were evaluated by enzyme- linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (PCR), respectively. Western blotting was utilized for assessing p65-NF-κB phosphorylation. Ransod kits evaluated the glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity in the cell lysates. Ultimately, the molecular docking approach was performed to investigate the biological activity of Quercetin against NF-κB pathway proteins and antioxidant enzymes. The findings revealed that quercetin significantly attenuated the expression and secretion of inflammatory mediators and p65-NF-κB phosphorylation in LPS-induced PBMCs. Additionally, quercetin dose-dependently improved the activities of SOD and GPx enzymes and decreased LPS-mediated oxidative stress in PBMCs. Moreover, quercetin has a considerable binding affinity to IκKb, the core element of the NF-κB pathway and the antioxidant enzyme SOD. The data show that quercetin plays a vital role in ameliorating inflammation and oxidative stress caused by LPS in PBMCs.

Abstract B38: Tetravalent bispecific antibodies specific for HER2 and disialoganglioside GD2 to engage polyclonal T cells for osteosarcoma therapy

Abstract Introduction: Osteosarcoma is the most common pediatric high-grade bone tumor. Although multimodal therapeutic approaches have significantly improved patient survival to more than 60%, the prognosis for patients with metastatic or relapsed disease remains dismal—an urgent unmet need. Its genomic complexity and higher mutational burden compared to other pediatric cancers should provide neoantigens as potential targets for T cell-based immunotherapy; however, these tumors are in general “cold,” with insufficient or inactive tumor-infiltrating lymphocytes. Ganglioside GD2 is a tumor-associated surface antigen expressed in a broad spectrum of pediatric malignancies, including neuroblastoma, brain tumors, Ewing’s sarcoma, rhabdomyosarcoma, and osteosarcoma, while being restricted in normal tissues. Another promising target, HER2 or ErbB2, is overexpressed in many aggressive malignancies, including pediatric medulloblastoma, nephroblastoma, osteosarcoma, and desmoplastic small round cell tumor (DSRT). Fully humanized tetravalent bispecific antibodies (BsAb) specific for human GD2 and CD3 (hu3F8-BsAb) or HER2 and CD3 (HER2-BsAb), built on an IgG(L)-scFv platform, have been developed (Oncoimmunology 2017; Can Immunol Res 2015). They induce rapid and quantitative T-cell homing to tumors, mediating potent T-cell dependent cytotoxicity (TDCC) against GD2 or HER2 expressing tumor cells, effecting cures of xenografts in SCID mice. In this study, we evaluated the in vitro and in vivo antitumor properties of these BsAbs in the treatment of osteosarcoma. Methods: Hu3F8-BsAb and HER2-BsAb were tested both in vitro and in vivo against a panel of osteosarcoma cell lines (RG143B, U2OS, CRL1427, HOS, and SaOS2). Using these cell lines, FACS analysis for surface antigen (GD2 and HER2) expression (MFI), sensitivity to TDCC (EC50), and antitumor activity in vivo were evaluated. Two in vivo tumor models with different effector routes were used to simulate different clinical situations: 1) subcutaneous (sc) tumor cells/sc effector peripheral blood mononuclear cells (PBMCs), and 2) sc tumor cells/intravenous (iv) PBMCs. In vivo tumor responses were measured by Peira TM900 imaging device or by bioluminescence. Results: The majority of osteosarcoma cell lines express GD2 and HER2 on their surface, and their in vitro sensitivity to TDCC (EC50) was inversely correlated with MFI of their respective antigen. Both hu3F8-BsAb and HER2-BsAb mediated potent TDCC against osteosarcoma cell lines. In vivo, both hu3F8-BsAb and HER2-BsAb exerted a significant antitumor effect compared to control BsAb (P=0.001). Conclusions: Hu3F8-BsAb and HER2-BsAb induced strong TDCC and had significant antitumor effect against osteosarcoma cell lines both in vitro and in vivo. Considering the limited therapeutic options currently available in advanced osteosarcomas, these results support their further clinical development as potential T cell-based immunotherapeutics. Citation Format: Jeong A. Park, Hong Xu, Irene Cheung, Nai-Kong V. Cheung. Tetravalent bispecific antibodies specific for HER2 and disialoganglioside GD2 to engage polyclonal T cells for osteosarcoma therapy [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B38.

Methods to evaluate cytotoxicity and immunosuppression of combustible tobacco product preparations.

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to increased susceptibility of smokers to microbial infections and tumor incidence. Ex vivo suppression of receptor-mediated immune responses in human peripheral blood mononuclear cells (PBMCs) treated with smoke constituents is an attractive approach to study mechanisms and evaluate the likely long-term effects of exposure to tobacco products. Here, we optimized methods to perform ex vivo assays using PBMCs stimulated by bacterial lipopolysaccharide, a Toll-like receptor-4 ligand. The effects of whole smoke-conditioned medium (WS-CM), a combustible tobacco product preparation (TPP), and nicotine were investigated on cytokine secretion and target cell killing by PBMCs in the ex vivo assays. We show that secreted cytokines IFN-γ, TNF, IL-10, IL-6, and IL-8 and intracellular cytokines IFN-γ, TNF-α, and MIP-1α were suppressed in WS-CM-exposed PBMCs. The cytolytic function of effector PBMCs, as determined by a K562 target cell killing assay was also reduced by exposure to WS-CM; nicotine was minimally effective in these assays. In summary, we present a set of improved assays to evaluate the effects of TPPs in ex vivo assays, and these methods could be readily adapted for testing other products of interest.

Elimination of Human T Cell Leukemia Virus Type-1-Infected Cells by Neutralizing and Antibody-Dependent Cellular Cytotoxicity-Inducing Antibodies Against Human T Cell Leukemia Virus Type-1 Envelope gp46

Human T cell leukemia virus type-1 (HTLV-1) is prevalent worldwide with foci of high prevalence. However, to date no effective vaccine or drug against HTLV-1 infection has been developed. In efforts to define the role of antibodies in the control of HTLV-1 infection, we capitalized on the use of our previously defined anti-gp46 neutralizing monoclonal antibody (mAb) (clone LAT-27) and high titers of human anti-HTLV-1 IgG purified from HAM/TSP patients (HAM-IgG). LAT-27 and HAM-IgG completely blocked syncytium formation and T cell immortalization mediated by HTLV-1 in vitro. The addition of these antibodies to cultures of CD8(+) T cell-depleted peripheral blood mononuclear cells (PBMCs) from HAM/TSP patients at the initiation of culture not only decreased the numbers of Tax-expressing cells and the production of HTLV-1 p24 but also inhibited the spontaneous immortalization of T cells. Coculture of in vitro-HTLV-1-immortalized T cell lines with autologous PBMCs in the presence of LAT-27 or HAM-IgG, but not an F(ab')2 fragment of LAT-27 or nonneutralizing anti-gp46 mAbs, resulted in depletion of HTLV-1-infected cells. A 24-h (51)Cr release assay showed the presence of significant antibody-dependent cellular cytotoxicity (ADCC) activity in LAT-27 and HAM-IgG, but not F(ab')2 of LAT-27, resulting in the depletion of HTLV-1-infected T cells by autologous PBMCs. The depletion of natural killer (NK) cells from the effector PBMCs reduced this ADCC activity. Altogether, the present data demonstrate that the neutralizing and ADCC-inducing activities of anti-HTLV-1 antibodies are capable of reducing infection and eliminating HTLV-1-infected cells in the presence of autologous PBMCs.

Neutralizing antibodies against human T cell leukemia virus type-I (HTLV-1) eradicate HTLV-1 in combination with autologous peripheral blood mononuclear cells via antibody-dependent cellular cytotoxicity while preventing new infection

In order to establish a basis for vaccine development against human T cell leukemia virus type-I (HTLV-1), we have evaluated the roles of anti-HTLV-1 neutralizing antibodies using a rat monoclonal antibody (mAb) against HTLV-1 envelope gp46 (LAT-27) and human polyclonal IgG purified from sera of HTLV-1-associated myelopathy (HAM) patients (HAM-IgG). LAT-27 and HAM-IgG completely blocked the HTLV-1-mediated syncytium formation and T-cell transformation in vitro. Interestingly, when these antibodies were added to the cultures of CD8+ T cell-depleted peripheral blood mononuclear cells (PBMCs) from HAM patients, proliferation of Tax-expressing T cells and HTLV-1 p24 production were blocked. In addition, co-culture of HTLV-1-immortalized T cells with autologous PBMCs in the presence of LAT-27 or HAM-IgG, but not F(ab)’2 fragment of LAT-27 or the other non-neutralizing anti-gp46 mAbs, resulted in eradication of Tax-expressing cells and the p24 production. A 51Cr release assay for 24 hours showed a significant killing of HTLV-1-infected T cells by autologous PBMCs in the presence of LAT-27 or HAM-IgG, but not F(ab)’2 fragment of LAT-27, in which depletion of CD16+ cells from the effector PBMCs significantly reduced the killing activity. Altogether, the present data demonstrated for the first time that anti-HTLV-1 gp46 neutralizing antibodies are capable of not only preventing new infection but also eliminating HTLV-1-infected cells in the presence of autologous PBMCs mainly via an antibody-dependent cellular cytotoxicity (ADCC) in vitro. Thus, a vaccine candidate that can elicit or boost anti-gp46 neutralizing antibody response may have a potential for prevention and therapy against HTLV-1 infection.

Immune modulation effects of concomitant temozolomide and radiation therapy on peripheral blood mononuclear cells in patients with glioblastoma multiforme

Concomitant radiation therapy (RT) and temozolomide (TMZ) therapy after surgery is the standard treatment for glioblastoma multiforme (GBM). Radiation and chemotherapy can affect the immune system with implications on subsequent immune therapy. Therefore, we examined the phenotype and function of peripheral blood mononuclear cells in 25 patients with GBM prior to and 4 weeks after treatment with RT-TMZ using multicolor flow cytometry, as well as in vitro CD4(+) regulatory T cell (T(reg)) suppressor and dendritic cell maturation assays. RT-TMZ induced significant lymphopenia, with a decrease in total CD4(+) T cells, but did not significantly change monocyte counts. The proportion of functional T(reg) cells increased after treatment, whereas their absolute numbers remained stable. There was also a measurable decrease in the proportion of CD8(+)CD56(+) and absolute number of CD3(-)CD56(+) effector cells. Posttherapy monocytes retained the ability to mature into dendritic cells. Treatment with RT-TMZ is associated with changes in regulatory and effector peripheral blood mononuclear cells that tilt the balance towards an immune suppressive state. This shift can affect the outcome of immune therapy following RT-TMZ treatment and should be considered in the design of future combination therapy regimens.

Daratumumab Directly Induces Human Multiple Myeloma Cell Death and Acts Synergistically with Conventional and Novel Anti-Myeloma Drugs

AbstractAbstract 3013Daratumumab is a novel fully human therapeutic CD38-specific monoclonal antibody (mAb) that is currently in phase I/II safety and dose finding clinical studies in MM. We recently demonstrated that daratumumab induces antibody dependent cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) against multiple myeloma (MM) cells (ASH Abstract #608, 2009). Significantly, daratumumab induces ADCC-mediated autologous lysis against MM patient cells. In addition, when cross-linked, daratumumab directly induces Ramos lymphoma cell death. We here studied whether daratumumab directly kills MM cells and whether daratumumab could be combined with other anti-MM drugs to further enhance its direct cytotoxicity. Direct daratumumab-induced MM cell death was determined using CellTiter-Glo luminescent cell viability assay and Annexin V/PI flow cytometry analysis, with or without goat anti-human IgG crosslinking. Following 48h incubation, daratumumab (0.1-10 μg/ml), when cross-linked, directly induced cytoxicity against dexamethasone (dex)-sensitive MM1S and dex-resistant MM1R cells, as evidenced by decreased cell viability in a dose-dependent manner. Importantly, cross-linked daratumumab increased caspase 3/7 activities in a dose-dependent fashion, as assessed by the Caspase-Glo® 3/7 luminescence assay. Furthermore, daratumumab upregulated Annexin V+ and Annexin V+/PI+ cells in freshly isolated CD138+ MM patient cells, from 7.7% to 20.6% and 10.9% to 15.4 %. Therefore, cross-linked daratumumab can directly trigger apoptosis of patient myeloma cells. Cell viability assay was further performed on MM1S cells when daratumumab (0.1, 1, 10 μg/ml) was combined with dex (0.5 and 1 μM) or bortezomib (2.5, 5, and 10 nM). Following 48–72h incubation with daratumumab, both dex and bortezomib synergistically inhibited MM cell viability, as determined by combination index (CI) < 0.5 at given combined concentrations of these drugs. Enhanced caspase 3/7 activation was also seen when daratumumab was combined with dex. To evaluate combination cytotoxicity induced by lenalidomide with daratumumab, peripheral blood mononuclear effector cells (PBMCs) from normal donors (n=2) were pretreated with lenalidomide (2 μM) for 3 days followed by daratumumab-mediated ADCC assays against MM1S cells. Using calcein-AM release measurements, lenalidomide-pretreated PBMCs further augmented daratumumab-induced MM1S cell lysis, whereas daratumumab-pretreated PBMCs did not alter ADCC. Taken together, our studies show that daratumumab directly induces MM cell death via activation of caspase 3/7 and daratumumab induced synergistic cytotoxicity with dex or bortezomib. Moreover, lenalidomide augments daratumumab-induced ADCC against MM cells. These results further support combination clinical trials of conventional and novel anti-MM drugs with daratumumab in MM. Disclosures:Weers:Genmab: Employment. Parren:Genmab: Employment. Richardson:Keryx Biopharmaceuticals: Honoraria. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Integrated preclinical and clinical development of mTOR inhibitors in pancreatic cancer

Background:The purpose of this work was to determine the efficacy of inhibiting mammalian target of rapamycin (mTOR) in pancreatic cancer preclinical models and translate preclinical observations to the clinic.Methods:Temsirolimus (20 mg Kg−1 daily) was administered to freshly generated pancreatic cancer xenografts. Tumour growth inhibition was determined after 28 days. Xenografts were characterised at baseline by gene expression and comparative genomic hybridisation. Patients with advanced, gemcitabine-resistant pancreatic cancer were treated with sirolimus (5 mg daily). The primary end point was 6-month survival rate (6mSR). Correlative studies included immunohistochemistry assessment of pathway expression in baseline tumours, drug pharmacokinetics (PKs), response assessment by FDG-PET and pharmacodynamic effects in peripheral-blood mononuclear cells (PBMCs).Results:In all, 4 of 17 xenografts (23%) responded to treatment. Sensitive tumours were characterised by gene copy number variations and overexpression of genes leading to activation of the PI3K/Akt/mTOR pathway. Activation of p70S6K correlated with drug activity in the preclinical studies. Sirolimus was well tolerated in the clinic, showed predictable PKs, exerted pathway inhibition in post-treatment PBMCs and resulted in a 6mSR of 26%. No correlation, however, was found between activated p70S6K in tumour tissues and anti-tumour effects.Conclusion:Sirolimus activity in pancreatic cancer was marginal and not predicted by the selected biomarker.

Rapid production of recombinant human IgG With improved ADCC effector function in a transient expression system

Rapid production of recombinant human IgG with improved antibody dependent cell-mediated cytotoxicity (ADCC) effector function is presented. The technique employs transient expression of IgG in suspension growing HEK-293F cells in the presence of the glycosidase inhibitor kifunensine. The procedure takes approximately 7 days, provided that expression plasmids encoding the IgG of interest are available. Kifunensine inhibits the N-linked glycosylation pathway of HEK-293F cells in the endoplasmatic reticulum, resulting in IgG with oligomannose type glycans lacking core-fucose. IgG1 transiently produced in kifunensine- treated HEK-293F cells has improved affinity for the FcgammaRIIIA molecule as measured in an ELISA based assay, and almost eightfold enhanced ADCC using primary peripheral blood mononuclear effector cells.

Lentiviral Engineered T Regulatory Cells Effectively Inhibit Lymphocytes Alloreactivity across Major HLA Barriers

Adoptive immunotherapy strategies enrolling T regulatory cells (Tregs) might have a great potential in modulating donor T cells alloreactivity after Hematopoetic Cell Transplant (HCT). In murine models of HCT Tregs were shown to promote engraftment and contribute controlling graft versus host disease (GVHD) while still not conclusive data are available on humans. Ex-vivo engineering conventional CD4+ T cells to over-express the transcription factor FOXP3 is an intriguing approach to overcome the main difficulty of obtaining large amount of Tregs for experimental studies. Reports of retrovirus-mediated expression of FOXP3 not consistently resulted in functional Tregs while, recently, a lentivirus-mediated strategy was successfully reported to result in homogeneous and stable expression of FOXP3. Lentiviral transduced Tregs were able to suppress a polyclonal proliferation of CD4 purified lymphocytes stimulated with soluble Ab anti-CD3. In our study we generated lentiviral engineered Tregs (eng-Tregs) and investigated their inhibitory effect on unselected lymphocytes alloreactivity across major HLA barriers. Within the bulk lymphocytes population we separately tracked the suppressive influence of eng-Tregs on both CD4+ and CD8+ T cells. To obtain eng-Tregs, CD4+ T cells were purified from healthy donors and transduced with a bidirectional lentiviral vector encoding for FOXP3 and the truncated Nerve Growth Factor Receptor (ΔNGFR). Prior to transduction CD4+ cells were activated for 72 hours with IL2 (100U/ml), IL7 (20ng/ml) and soluble Ab anti-CD3 (200 ng/ml, only IL2 was added to the culture medium after transduction.The lentiviral transduction efficiency ranged from 8 to 25%, ΔNGFR+ T cells were positively selected and tested for their ability to suppress a mixed lymphocyte reaction across major HLA barriers. Effector peripheral blood mononuclear cells (PBMC) were collected from the same donors from whom eng-Tregs were generated. Effector PBMC were stained with CFSE in oder to separately track the alloreactive proliferation of both CD8+ and CD4+ subsets of T cells. Eng-Tregs were added on day 0 and HLA-mismatched irradiated PBMC were used as stimulators; both eng-Tregs and irradiated stimulators were used in a 1:1 ratio with the effectors. No cytokines or additional soluble stimulators were added to the MLR culture medium. The alloreactive proliferation of T cell subsets was determined by evaluating the logarithmic decrease of CFSE fluorescence intensity. The flow cytometry analysis on day +7 showed that alloreactive proliferation of both CD4+ and CD8+ effector cells was significantly inhibited (>75%) by the addition of eng-Tregs compared to controls. In order to rule out a possible role played by the naturally present Tregs (nat-Tregs), the effectors were depleted of the CD4+CD25high subpopulation before the MLR started. The observed alloreactive proliferation was higher after the depletion of nat-Tregs but still it could be significantly inhibited by the addition of eng-Tregs. Eng-Tregs did not significantly expanded when cultured in vitro (up to 2 weeks) with IL2 (100U/ml) but maintained a stable expression of the transgene and retained their suppressive capacity. Our data show that lentiviral engineered Tregs can efficiently down-modulate both CD4+ and CD8+ T cell alloreactivity across major HLA barriers. The observed independence from the presence of nat-Tregs might be important in future experimental HCT settings where the adoptive infusion of eng-Tregs might encounter a great variability in the number and activity of recipient's nat-Tregs. The possibility of transducing a potentially unlimited number of CD4+ cells makes this strategy appealing for future pre-clinical studies to control GVHD in HCT settings.

Characterisation of the new EpCAM-specific antibody HO-3: implications for trifunctional antibody immunotherapy of cancer

Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein that is frequently overexpressed in a variety of carcinomas. This pan-carcinoma antigen has served as the target for a plethora of immunotherapies. Innovative therapeutic approaches include the use of trifunctional antibodies (trAbs) that recruit and activate different types of immune effector cells at the tumour site. The trAb catumaxomab has dual specificity for EpCAM and CD3. In patients with malignant ascites, catumaxomab significantly increased the paracentesis-free interval, corroborating the high efficacy of this therapeutic antibody. Here, we characterised the monoclonal antibody (mAb) HO-3, that is, the EpCAM-binding arm of catumaxomab. Peptide mapping indicated that HO-3 recognises a discontinuous epitope, having three binding sites in the extracellular region of EpCAM. Studies with glycosylation-deficient mutants showed that mAb HO-3 recognised EpCAM independently of its glycosylation status. High-affinity binding was not only detected for mAb HO-3, but also for the monovalent EpCAM-binding arm of catumaxomab with an excellent KD of 5.6 × 10−10 M. Furthermore, trAb catumaxomab was at least a 1000-fold more effective in eliciting the eradication of tumour cells by effector peripheral blood mononuclear cells compared with mAb HO-3. These findings suggest the great therapeutic potential of trAbs and clearly speak in favour of EpCAM-directed cancer immunotherapies.

Recombinant gp120 Vaccine-Induced Antibodies Inhibit Clinical Strains of HIV-1 in the Presence of Fc Receptor-Bearing Effector Cells and Correlate Inversely with HIV Infection Rate

Nonneutralizing Abs may play a role in protecting animals and humans from lentiviral infections. We explored the Ab-dependent, cell-mediated virus inhibition (ADCVI) Ab response to recombinant gp120 (rgp120) vaccination in sera from 530 participants in the Vax 004 trial. Serum ADCVI activity was measured against a clinical R5 strain of HIV-1 using peripheral blood mononuclear effector cells from healthy donors. The level of vaccine-induced ADCVI activity correlated inversely with the rate of acquiring HIV infection following vaccination, such that for every 10% increase in ADCVI activity, there was a 6.3% decrease in the hazard rate of infection (p=0.019). Some vaccinated individuals also mounted an ADCVI response against two other clinical R5 strains of HIV-1. However, ADCVI activity correlated poorly with neutralizing or CD4-gp120-blocking Ab activity measured against laboratory strains. Finally, the degree to which the ADCVI Ab response predicted the rate of infection was influenced by polymorphisms at the FcgammaRIIa and FcgammaRIIIa gene loci. These data indicate that rgp120 vaccination can elicit Abs with antiviral activity against clinical strains of HIV-1. However, such activity requires the presence of FcR-bearing effector cells. Our results provide further evidence that ADCVI may play a role in preventing HIV infection.

Impaired cytolytic activity in peripheral blood T cells from renal cell carcinoma patients

Classic T lymphocyte cytotoxicity is mediated through the T cell receptor (TCR). Defects in TCR signal transduction and cytolytic activity have been reported in tumor infiltrating T lymphocytes. We hypothesized that impaired cytotoxicity occurs in peripheral blood T cells from renal cell carcinoma (RCC) that can be reversed by exposure to rhIL-2. Peripheral blood mononuclear cells (PBMC) from 29 RCC patients and 29 healthy volunteers were isolated and cultured in the absence or presence of 10 IU/ml rhIL-2. A redirected cytotoxicity assay that requires TCR signal transduction was used with chromium-labeled P815 target cells, effector PBMC and anti-CD3 antibody. Target cell lysis was measured in “lytic units” (LU). Mean LU from RCC patients was lower than that of healthy volunteers (105.8 LU vs. 194.6 LU, P = 0.025). Exposure to rhIL-2 increased T cell-mediated lysis in both groups. Disruption of T cell cytotoxicity in RCC patients can be overcome by exposure to rhIL-2.

Influence of menstrual cycle on NK activity

Natural killer (NK) cells are CD3 − CD56 + and/or CD16 + cytotoxic lymphocytes that mediate first-line defense against various types of target cells without prior immunization. To assess the effect of the menstrual cycle and gender on NK activity we evaluated 30 healthy women (mean age 28.1 years, range 21–39) in follicular and luteal phases, 29 postmenopausal women (mean age 58.8 years, range 42–72) and 48 healthy men (mean age 31.6 years, range 21–40). In a flow cytometric test of NK activity, peripheral blood mononuclear effector cells were mixed with K562 targets cells labeled with DiO (3,3′-dioctadecyloxacarbocyanine perchlorate) at effector:target cell ratios of 40, 20, 10 and 5:1. Dead cells were stained with propidium iodide and results were expressed as lytic units per 10 7 cells. In addition, progesterone levels were determined in the luteal phase of the menstrual cycle of healthy women by a chemiluminescence assay. Our results showed that (1) NK cytotoxicity was higher in the follicular than in the luteal phase of the menstrual cycle ( P<0.0001); (2) postmenopausal women and men showed NK activity similar to women in the folicular phase but higher than women in the luteal phase of the menstrual cycle ( P<0.05); and (3) there was no correlation between NK activity and levels of progesterone. The data suggest that progesterone does not influence NK activity directly and that other factors may explain the reduction of NK activity in the luteal phase of the menstrual cycle.

Use of recombinant vaccinia to restimulate antigen specific human peripheral blood cytotoxic T lymphocytes

A simple method is described for using recombinant vaccinia virus for restimulating human peripheral blood mononuclear cells (PBMC) to generate antigen specific CD8 + αβ cytotoxic T cell (CTL) effector populations. Effector PBMC were restimulated in vitro with a subpopulation of PBMC, which had been infected with recombinant vaccinia at very low multiplicities of infection in the absence of serum. This protocol avoided the vaccinia mediated overt cytopathic effects on the effector PBMC and generated bulk CTL cultures, which could be used for the identification of epitopes recognised by antigen specific CTL.

Profile of cytokine production in mixed kidney lymphocyte culture.

Kidney cells are an important source of immunoregulatory molecules that regulate cell-to-cell interactions, which is the key step in the generation of the immunoresponse to alloantigens. In this study we identified the cytokines that are produced by both lymphoid cells and kidney cells when coincubated in mixed kidney lymphocyte cultures (MKLC). The capacity of kidney cells to stimulate the proliferation of effector allogeneic lymphocytes was assayed by incubating irradiated kidney cells and lymphocyte. The cytokine secretion profile in MKLC was investigated by incubating monolayers of kidney cells with effector peripheral blood mononuclear cells (PBMC). The culture supernatants were harvested on days 1, 2, 3, 4, 5, 6, 7, and 8 and assayed for IL-1beta, IL-2, IL-6, and TNF alpha using an ELISA. Kidney cells, in comparison to PBMC stimulator cells were poor stimulators of the alloproliferation even when HLA expression was increased by IFN gamma treatment. Compared to lymphocyte or kidney cells incubated alone, MKLC induced a considerable stimulation of cytokine production. This increase in cytokine production was observed essentially for IL-2 and IL-6 (at day 3, a 10-fold increase in IL-2 and a 5-fold increase in IL-6). This study provided evidence that target kidney cells and effector lymphocyte interactions generate a number of cytokines such as IL-1beta, IL-2, IL-6, or TNF alpha. These cytokines are known to modulate allo-proliferation and generation of cytotoxic T lymphocytes (CTL).

Enhancing Effects of Interleukin 2-Treated Peripheral Blood Mononuclear Cells on Subsequent B Cell Differentiation

Despite an extensive literature dealing with IL2-induced cytolytic activity, noncytotoxicity-related effects of IL2 on peripheral blood mononuclear cells (PBMC) or T cell function have received less attention. We have focused on the effects of irradiated, IL2-activated PBMC (PBMC*rIL2) on anti-CD3- and formalin-fixed heat-killed Staphylococcus aureus-induced polyclonal B cell differentiation in secondary cultures. PBMC*rIL2 act directly on B cells and cross major histocompatibility complex barriers to augment polyclonal B cell differentiation as measured by plaque-forming cell (PFC) generation. These effects are preferentially mediated by T (both CD4+ and CD8+) cells, and physical contact between effector PBMC*rIL2 and target B cells is not absolutely required for enhanced PFC generation. PBMC*rIL2 must be present for the initial 24 hr of the secondary cultures, indicating that some soluble B cell differentiation factor rapidly released by PBMC*rIL2 mediates the PFC-enhancing effect. Of IL2, IL4, IL5, IL6, IL10, IFN-γ, and TNF-α, only IFN-γ mRNA is appreciably and reproducibly increased in irradiated, IL2-activated T cells (T cells*rIL2). Nevertheless, exogenous rIFN-γ cannot mimic and anti-IFN antibodies cannot block the PFC-enhancing effects of T cells*rIL2, indicating that some unidentified soluble factor(s) apart from or in addition to IFN-γ is involved. IL2-induced effects on T cell noncytolytic function may help explain certain observed immune anomalies in IL2-treated patients, and a better understanding of the IL2-induced effects on T cell noncytolytic function may have ramifications for autoimmune diseases such as SLE.

Elevated prostaglandin E2 production by monocytes is responsible for the depressed levels of natural killer and lymphokine-activated killer cell function in patients with breast cancer

Human natural killer (NK) cells mediate spontaneous cytotoxicity against tumor cells and represent the main precursors of lymphokine-activated killer (LAK) cell activity. A comparison of some aspects of NK and LAK cell activity was undertaken in 85 preoperative patients with breast cancer and 75 healthy donors. NK cell activity (tested in 18-hour cultures of effector peripheral blood mononuclear cells [PBMC] with K562 or MOLT-4 tumor target cells) was significantly diminished in these patients as it was the fully mature LAK cell activity (i.e., interleukin-2 (IL-2)-induced cytotoxicity in PBMC) against NK resistant target cells. Using immunoenzymatic methods we showed that the reduced NK cell activity was due to abnormally high levels of prostaglandin E2 (PGE2) produced by monocytes in culture. PGE2 was found to suppress the production of IL-2 in these cultures. Removal of monocytes from PBMC restored to almost normal levels the deficient NK and LAK cell activity in patients with breast cancer and was also associated with a normalization in the levels of PGE2 and IL-2. Indomethacin and gamma-interferon (IFN-gamma) increased the NK and LAK cell activity in these patients up to the levels of healthy donors. When highly purified CD56+ cells (obtained by an immunomagnetic isolation technique) were used as effector cells, no differences in LAK cell activity could be noticed between healthy donors and patients with cancer. FACS and northern blot analyses demonstrated a PGE2-mediated down-regulation of IL-2 receptor (IL-2R) expression on CD56+ cells that correlated with reduced LAK cell activity. This inhibitory effect of PGE2 was noticeable in long-term LAK cultures and was abrogated in the presence of IFN-gamma or indomethacin. This study may have important implications in the potentiation of NK and LAK cell activity for immunotherapeutic protocols in patients with breast cancer.

Suppressive effects of pentoxifylline on natural killer cell activity.

The methylxanthine derivative pentoxifylline, widely used as a hemorrheologic agent in the treatment of peripheral vascular disease, is now being evaluated for potential applications in patients with cancer. Recent studies have shown that pentoxifylline can modulate a number of neutrophil functions at in vitro concentrations of at least 50 micrograms/ml. Using a standard51chromium-release assay, we studied the suppressive effects of pentoxifylline on natural killer (NK) cell activity and found that pentoxifylline, at concentrations of 50 and 100 micrograms/ml, suppressed the in vitro NK cell activity of healthy volunteers by 25% and 75%, respectively. Postreaction supernatants from chromium-release studies were then assayed for prostaglandin E2 (PGE2) and tumor necrosis factor-alpha (TNF-alpha) by using enzyme-linked immunosorbent assay. Concentrations of both PGE2 and TNF-alpha were increased by more than five times in those assay wells containing pentoxifylline. Moreover, the addition of 1 microgram/ml indomethacin to the NK assay system containing pentoxifylline, completely inhibited PGE2 production and abrogated the pentoxifylline-induced NK suppression. The addition of PGE2 (1 x 10(-6) mol/L) to the assay system suppressed NK activity, whereas addition of 1 or 10 ng/ml TNF-alpha did not. Theophylline, another methylxanthine, failed to suppress NK activity like pentoxifylline at equimolar concentrations. Our studies provide the first evidence that concentrations of pentoxifylline of at least 50 micrograms/ml suppressed NK cell function by inducing PGE2 synthesis from effector peripheral-blood mononuclear cells.

Effects of low-dose mafosfamid on the lymphokine-activated killer cell activity of peripheral blood mononuclear cells determined by a clonogenic microassay

The effects of mafosfamid on lymphokine-activated killer (LAK) cells were examined, using a newly developed clonogenic microassay with tumor target cells instead of the common 51Cr release. When nonadherent peripheral blood mononuclear effector cells were exposed to 1 x 10(-9) or 1 x 10(-8) M mafosfamid 1 h prior to activation with interleukin 2 (IL-2), LAK-cell activity increased, resulting in fewer colonies of human KB squamous carcinoma cells than without mafosfamid treatment. In contrast, when effector cells were exposed to mafosfamid after IL-2 activation LAK-cell activity was inhibited. Notably, the in vitro immunomodulatory effects of mafosfamid were detected at concentrations 100-1000 times below those shown to be cytotoxic. Although the mechanisms remain to be elucidated, our results might be an in vitro representation of the known in vivo immunostimulation of low doses of cyclophosphamide.