Polyclonal T-cell Activation Research Articles

Inherited Hemophagocytic Lymphohistiocytosis (HLH)

Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal immune disorder characterized by uncontrolled lymphocyte- and macrophage-activation. The resulting hypercytokinemia and cell infiltration of organs lead to the clinical and laboratory features of HLH. Viral infections and other triggers can induce both, inherited and acquired forms of HLH. Disease-causing mutations in the genes encoding perforin (PRF1, FHL2), munc13-4 (UNC13D, FHL3), syntaxin 11 (STX11, FHL4), and munc18-2 (UNC18-2/STXBP2, FHL5) have been previously identified in Familial Hemophagocyic Lymphohistiocytosis (FHL), whereas mutation in RAB27A and LYST account for Griscelli syndome type 2 and Chediak-Higashi syndrome, respectively. These genes all encode proteins which are involved in the cytotoxic activity of lymphocytes. The inability of activated cytotoxic cells to clear antigen-presenting targets results in sustained immune stimulation, likely accounting for the unremitting polyclonal CD8 T-cell activation and hyperimmune reaction which characterizes FHL. Treatment of HLH consists of elimination of the trigger and immunosuppressive treatment in order to induce remission from the uncontrolled inflammation. Allogeneic hematopoietic stem cell transplantation can be indicated in the inherited forms of HLH.

Indirect inhibition of in vivo and in vitro T-cell responses by intravenous immunoglobulins due to impaired antigen presentation

AbstractSeveral clinical studies done with intravenous immunoglobulin (IVIg)–treated autoimmune patients as well as several in vitro studies have revealed that IVIg can reduce polyclonal T-cell activation and modify their cytokine secretion pattern. However, their effect on (auto)antigen-specific T-cell responses has never been addressed directly. In the present work, we used an in vivo model of induction of antigen-specific T-cell responses and an in vitro antigen presentation system to study the effects of IVIg on T-cell responses. The results obtained showed that IVIg inhibited both the in vivo and in vitro antigen-specific T-cell responses but that this effect was the indirect consequence of a reduction in the antigen presentation ability of antigen-presenting cells. The inhibitory effect of IVIg was FcγRIIb-independent, suggesting that IVIg must interfere with activating FcγRs expressed on antigen-presenting cells to reduce their ability to present antigens. Such inhibition of T-cell responses by reducing antigen presentation may therefore contribute to the well-known anti-inflammatory effects of IVIg in autoimmune diseases.

Suppressive activity rather than frequency of FoxP3 + regulatory T cells is essential for CA-125–specific T-cell activation after abagovomab treatment

The results of several clinical trials have clearly demonstrated the potential of the anti-idiotype (anti-Id) vaccine abagovomab to induce cancer antigen 125 (CA-125)-specific immunity in ovarian cancer patients. Because of the central role of regulatory T cells (Tregs) in tumor immunology, we analyzed the frequency and suppressive activity of CD25 +FoxP3 + Tregs in 16 patients treated with abagovomab. During vaccination, mean frequencies of peripheral Treg with a CD4 +CD25 +FoxP3 + CD127 − phenotype were enhanced but returned to baseline levels in the follow-up phase. Despite increasing Treg counts, the suppressive activity of Tregs was diminished in a subset of patients treated with abagovomab. Reduced Treg activity was associated with increasing polyclonal and CA-125–specific T-cell proliferation in these patients. Interestingly, CA-125–specific T-cell activation could not be further improved by Treg depletion in vitro, as CA-125 induced a suppressive CD4 +CD25 +FoxP3 + CD127 − T cell subset derived from the originally Treg-depleted T-cell fraction. These CA-125–induced Tregs (iTregs) efficiently blocked polyclonal and tumor-specific T-cell activation. Further elimination of iTregs resulted in detectable CA-125–specific T-cell responses in a subset of patients. Based on our results, the suppressive potential rather than the frequency of natural and CA-125–induced Tregs is an important issues to consider for refinement of current anti-Id vaccination.

An Alkaloid Extract of Evanta, Traditionally Used as Anti‐leishmania Agent in Bolivia, Inhibits Cellular Proliferation and Interferon‐γ Production in Polyclonally Activated Cells

Traditional medicine and scientific studies have shown that the raw extract of Evanta [Galipea longiflora, Angostura longiflora (Krause) Kallunki] exhibits anti-leishmanial activity. We hypothesized that the healing observed when using this plant might not only be due to the direct action on the parasite, but possibly to a parallel effect on the host immune response to the parasite involved in the healing process. We show here that an alkaloid extract of Evanta (AEE) directly killed the parasite already at a dose of 10 microg/ml, but at this low concentration, AEE did not have a major effect on viability and proliferation of eukaryotic cells. The whole extract was also found to be stronger than 2-phenylquinoline, the most prominent alkaloid in AEE. AEE was not directly stimulating B or T cells or J774 macrophages. However, it interfered with the activation of both mouse and human T cells, as revealed by a reduction of in vitro cellular proliferation and interferon-gamma (IFN-gamma) production. The effect was more evident when the cells were pretreated with AEE and subsequently stimulated with the polyclonal T-cell activators Concanavalin A and anti-CD3. Taken together, our results suggest that Evanta have a direct leishmanicidal effect and due to the effect on IFN-gamma production it might contribute to control the chronic inflammatory reaction that characterize Leishmania infection pathology, but in vivo studies are necessary to corroborate this finding.

A Pilot Trial of CTLA-4 Blockade with Human Anti-CTLA-4 in Patients with Hormone-Refractory Prostate Cancer

Blockade of the T-cell inhibitory receptor CTL-associated antigen-4 (CTLA-4) augments and prolongs T-cell responses and is a strategy to elicit antitumor immunity. The objectives of this pilot study were to establish the pharmacokinetic and safety profile for a single dose of 3 mg/kg of the anti-CTLA-4 antibody Ipilimumab (MDX-010, BMS-734016) and to assess if this therapy resulted in prostate-specific antigen (PSA) modulation and the development of polyclonal T-cell activation and/or clinical autoimmunity in patients with hormone-refractory prostate cancer treated with Ipilimumab. Patients with metastatic hormone-refractory prostate cancer received a single 3 mg/kg i.v. dose of Ipilimumab. Serologic measures of autoimmunity were obtained, and T-cell activation was evaluated by flow cytometry. Pharmacokinetic sampling of plasma for MDX-CTLA-4, PSA measurement, and diagnostic imaging were also undertaken. Fourteen patients were treated: 12 patients received a single dose of Ipilimumab, and 2 patients were re-treated with a second dose upon PSA progression. Two patients showed PSA declines of > or =50%. Treatment was well tolerated with clinical autoimmunity limited to one patient who developed grade 3 rash/pruritus requiring systemic corticosteroids. The mean +/- SD Ipilimumab terminal elimination half-life was 12.5 +/- 5.3 days. A single dose of 3 mg/kg Ipilimumab, an anti-CTLA-4 antibody, given to patients with prostate cancer is safe and does not result in significant clinical autoimmunity. PSA-modulating effects observed warrant further investigation.

Human Bladder Carcinoma is Dominated by T-Regulatory Cells and Th1 Inhibitory Cytokines

Immunotherapy has faced limited success, although many solutions have been proposed. Recently regulatory T cells have made a comeback in the immunological arena and the role of these cells in patients with cancer is in focus. It is under evaluation whether the immunological status of patients with cancer may affect their sensitivity to immunotherapy. We are developing immunostimulating gene therapy for treating bladder cancer. In this study we constructed an immunological profile of patients with bladder carcinoma to understand which obstacles must be circumvented. Biopsies and blood were used to identify immune cell populations by FACS(R), histochemistry and proliferation assays, and cytokine production by polymerase chain reaction. Results indicate that bladder carcinoma is a Tr1 dominated tumor, as shown by the infiltration of T-regulatory cells expressing FOXP3, and the presence of tumor necrosis factor-beta and interleukin-10 mRNA copies. We further noted that circulating patient T cells were unresponsive to polyclonal T-cell activation compared to healthy donor cells. Moreover, CD4+CD25+ T cells were increased in patient blood and could suppress the expansion of allogeneic T cells from healthy donors. Patients with bladder carcinoma show an immunosuppressive regulatory profile, including nonresponsive T cells. Clinical protocols able to effectively counteract these mechanisms are warranted.

Activation of T Cells by Bispecific Single-Chain Construct MT103 (MEDI-538) Is Strictly Target Cell-Dependent.

Background: Bispecific antibodies have been extensively studied in vitro and in vivo for their use in redirected tumor cell lysis. A particular challenge of bispecific antibody constructs recognizing the CD3 signaling complex is to achieve a controlled polyclonal activation of T-cells that, ideally, is entirely dependent on the presence of target cells. If this is not the case, systemic production of inflammatory cytokines and secondary endothelial reactions may occur as side effects, as are observed with the murine anti-human CD3e antibody OKT-3 (muromab, Orthoclone®). Here we present evidence that MT103 (or MEDI-538), a bispecific single chain antibody of the BiTE class that targets CD19 and CD3, induces T-cell activation exclusively in the presence of target cells.Material and methods: Peripheral blood mononuclear cells from healthy donors were prepared by Ficoll density centrifugation. PBMC were incubated for 24 hours with MT103 in presence or absence of specific target cells. Target cell lysis was determined by measurement of adenylate kinase activity released from lysed cells. De novo expression of activation markers CD69 and CD25 on T-cells was assessed by flow cytometry using directly conjugated monoclonal antibodies, and the concentration of cytokines in the supernatant was determined by a commercial FACS-based bead array.Results: MT103 was analyzed for conditional T-cell activation. In the presence of target-expressing cell lines, low picomolar concentrations of MT103 were sufficient to stimulate a high percentage of peripheral human T-cells to express cytokines and surface activation markers, to enter into the cell cycle and to induce redirected lysis of target cells. However, in the absence of target cells, the BiTE molecules no longer detectably activated human T-cells even at concentrations exceeding the ED50 for redirected lysis and conditional T-cell activation by more than five orders of magnitude.Conclusion: Our data show that T-cell activation by MT103 is highly conditional in that it is strictly dependent on the presence.

Reduced susceptibility to dextran sulphate sodium‐induced colitis in the interleukin‐2 heterozygous (IL‐2+/–) mouse

Summary Mice homozygous for an inactivation of the interleukin-2 (IL-2) gene develop a T-cell dependent colitis. Heterozygous (IL-2+/-) mice are clinically healthy but have been shown to express reduced levels of IL-2 in the colon. Splenocytes from the IL-2+/- mice had a poorer proliferative response to polyclonal T-cell activation and these mice have reduced numbers of intestinal regulatory T cells (CD4+ CD25+ cells) when compared to wild type mice. When exposed to dextran sulphate sodium (DSS) IL-2+/- mice showed a markedly reduced susceptibility to DSS-induced colitis. While DSS treatment caused a marked increase in both CD4+ and CD8+ colonic T cells expressing increased levels of IL-2, IL-4, and IL-10 in wild type mice none of these changes were seen in IL-2+/- mice. On the contrary, cytokine expression in intestinal T cells of IL-2+/- mice was actually reduced after DSS treatment. These results suggest that reduced levels of IL-2 leads to attenuated activation and function of intestinal T cells in IL-2+/- mice and a failure to react adequately to DSS exposure.

Preferential retroviral-mediated transduction of EBV- and CMV-specific T cells after polyclonal T-cell activation.

Graft-versus-host disease, resulting from the T cells present in allogeneic hematopoietic stem cell (HSC) inoculums, can potentially be treated if a suicide gene has been introduced into the donor T cells. However, the diversity and functionality of the transfused T-cell population, including EBV- (EBV-T) and CMV-specific (CMV-T) CD8+ T cells, which are particularly important for immunosuppressed individuals undergoing HSC transplants, are often modified by the gene transfer protocol. Here, we show that following polyclonal T-cell activation, EBV-T and CMV-T cells are preferentially transduced by oncoretroviral vectors, as compared to the bulk CD8+ T-cell population. This preferential transduction is associated with higher surface levels of PiT-2, the receptor for the amphotropic envelope with which the virions are pseudotyped. Moreover, EBV-T and CMV-T cells proliferate more extensively as compared to bulk CD8+ T cells. Thus, retroviral-mediated transduction can be biased toward a given antigenic specificity, even under conditions of polyclonal stimulation.

Inhibition of cytokine production by methotrexate. Studies in healthy volunteers and patients with rheumatoid arthritis.

To analyse whether the beneficial effects of methotrexate in rheumatoid arthritis (RA) could be due to inhibition of inflammatory cytokine production. Cytokine production was studied using whole blood (WB) and mononuclear cells (MNC) of healthy volunteers and RA patients. Cultures were stimulated with either bacterial products such as lipo-oligosaccharide (LOS) or Staphylococcus aureus Cowan I (SAC) to activate monocytes or with monoclonal antibodies to CD3 and CD28 to induce polyclonal T-cell activation. We analysed the effect of methotrexate on cytokine production in these systems. We showed that methotrexate inhibits production of cytokines induced by T-cell activation. Among the cytokines inhibited were interleukin 4 (IL-4), IL-13, IFN gamma, tumour necrosis factor-alpha (TNF alpha) and granulocyte-macrophage colony-stimulating factor. Inhibition was seen at concentrations easily achieved in plasma of RA patients taking the drug. IL-8 production was hardly influenced by methotrexate. Furthermore, inhibition was dependent on the stimulus; IL-6, IL-8, IL-1 beta and TNF alpha production induced by LOS or SAC was only slightly decreased by methotrexate. The addition of folinic acid or thymidine and hypoxanthine reversed the inhibitory effects of methotrexate on cytokine production. Concentrations of methotrexate required for inhibition varied between donors. Oral intake of 10 mg methotrexate by RA patients led to marked inhibition of cytokine production in blood drawn after 2 h. Methotrexate turns out to be an efficient inhibitor of cytokine production induced by T-cell activation in freshly drawn blood. This is due to inhibition of the de novo synthesis of purines and pyrimidines. Cytokines produced by monocytes are hardly affected by methotrexate.

Immunological Responses Are Not Abnormal in Symptomatic Gulf War Veterans

The underlying etiology and pathogenesis of Gulf War veterans' illnesses continue to be under intense investigation. Reports have suggested the basis for these illnesses may be an altered immune system, but compelling evidence is lacking. We sought to determine whether in vitro immune responses were abnormal in symptomatic Gulf War veterans relative to matched controls. A randomized case-control study was conducted by blinded comparison of laboratory measures of in vitro immune responses in blood samples obtained from veterans in an outpatient facility of a Veterans Affairs medical center. Symptomatic Gulf War veterans with otherwise undefined illnesses (52 symptomatic subjects), asymptomatic Gulf War veterans (31 asymptomatic controls), and veterans who had applied for disability compensation and had not participated in the Gulf War (21 disability controls) represented the volunteer sample. In vitro cellular and humoral immune responses were measured to detect functional abnormalities in antigen presenting cells (autologous mixed leukocyte reactions and expression of interleukin (IL)-1beta, IL-6, IL-10, and tumor necrosis factor-alpha); T cells (lymphocyte proliferation using the polyclonal T-cell activators phytohemagglutinin and Concanavalin A; primary immune responses in allogeneic mixed leukocyte reactions; secondary immune response using the recall antigens tetanus toxoid, Candida albicans, and anthrax vaccine; and soluble IL-2 receptor expression); type-1 T-helper cells (gamma interferon expression); type-2 T-helper cells (IL-4 and IL-10 expression); and B cells (polyclonal B-cell activator pokeweed mitogen-induced immunoglobulin production). In general, immune response measures did not differ significantly between groups. Heightened responses observed in the disability control group (sporadically greater responses to one mitogen and two antigens) and the Gulf War participation control group (greater recall responses to anthrax vaccine) did not suggest impaired immune cell function in symptomatic veterans when compared with controls. We conclude that in vitro immunological responses are not abnormal in symptomatic Gulf War veterans.

Crystal structure and conformation of linomide [N-methyl-N-phenyl-1,2-dihydro-4-hydroxy-1 -methyl-2-oxo-quinoline-3-carboxamide]: A novel immunomodulator

Linomide [N-methyl-N-phenyl-1,2-dihydro-4-hydroxy-1-methyl-2-oxo-quinoline-3-carboxamide] is a new immunomodulator from Phamacia Laboratories, Helsingborg, Sweden. Linomide (LS-2616) has delayed hypersensitivity in rat skin, potentiates mouse natural killer cell activity, and stimulates polyclonal T-cell activation. It has been shown that LS-2616 abolishes the effects of continuous cyclosporine treatment in experimental models. Crystal structure of LS-2616 has been undertaken as a first step in the establishment of a possible structure–function relationship for the drug. Crystals of LS-2616 (C18H16N2O3) obtained from methylene chloride are monoclinic, of space group P21/a with the following crystallographic parameters: a = 8.793(1) A, b = 22.349(3) A, c = 7.808(1) A, β = 92.96(1)°, V = 1532.3(6) A3, ρobs = 1.34 Mg/m3, ρcalc = 1.337 Mg/m3, and Z = 4. The structure was solved with CAD-4 data using MULTAN programs and refined to a final R value of 0.041. The N-methyl carboxamide is in the cis configuration and is turned away from the quinoline ring by 87°. FK506 is a novel 23-membered macrolide lactone which is currently used for bone marrow and organ transplantations. The crystal structure of FK506 has been published. The absolute configuration of FK506 was established as a cis-amide which contains a L-pipecolic acid moiety. Preliminary modeling studies of FK506 with LS-2616 revealed that this cis conformation for the N-methyl carboxamide leads to a lower binding energy than the corresponding trans conformation. The plane of the phenyl group is inclined by 95° to the carboxamide plane. The molecule has the familiar herring-bone type of packing, characteristic of the polyphenyl molecules, stabilized by O–H ⋅ ⋅ ⋅ O and C–H ⋅ ⋅ ⋅ O hydrogen bonds involving the hydroxyl group and the ketone substituents on the quinoline ring and the N-methyl group of the carboxamide.

Bacterial infections and atopic dermatitis.

Bacterial infections and atopic dermatitis.

Suppression of antigen-specific lymphocyte activation in modeled microgravity.

Various parameters of immune suppression are observed in lymphocytes from astronauts during and after a space flight. It is difficult to ascribe this suppression to microgravity effects on immune cells in crew specimens, due to the complex physiological response to space flight and the resultant effect on in vitro immune performance. Use of isolated immune cells in true and modeled microgravity in immune performance tests, suggests a direct effect of microgravity on in vitro cellular function. Specifically, polyclonal activation of T-cells is severely suppressed in true and modeled microgravity. These recent findings suggest a potential suppression of oligoclonal antigen-specific lymphocyte activation in microgravity. We utilized rotating wall vessel (RWV) bioreactors as an analog of microgravity for cell cultures to analyze three models of antigen-specific activation. A mixed-lymphocyte reaction, as a model for a primary immune response, a tetanus toxoid response and a Borrelia burgdorferi response, as models of a secondary immune response, were all suppressed in the RWV bioreactor. Our findings confirm that the suppression of activation observed with polyclonal models also encompasses oligoclonal antigen-specific activation.

Azodicarbonamide inhibits T-cell responses in vitro and in vivo.

Azodicarbonamide was recently identified as a new anti-HIV agent that targets the zinc finger domains of the HIV-1 NCp7 nucleocapsid protein. Here, we demonstrate that azodicarbonamide inhibits in a dose-dependent manner the responses of purified human CD4+ T lymphocytes stimulated either by monoclonal antibodies against CD3 and CD28 or by allogeneic dendritic cells. These suppressive effects involve a direct action on the calcium mobilization machinery, as azodicarbonamide strongly inhibited the calcium influx induced either by antibodies against CD3 and CD28 or the chemokine RANTES, as well as by thapsigargin, an activator of depletion-activated calcium channels. In vivo, administration of azodicarbonamide into mice blunted their response to polyclonal T-cell activation induced by the injection of monoclonal antibody against CD3 and resulted in delayed rejection of skin allografts. In addition to its anti-HIV properties, azodicarbonamide is a new immunosuppressive agent that might have therapeutic applications in T cell-mediated inflammatory disorders.

The clinician's choice of antibiotics in the treatment of bacterial skin infection

The development of modern antibiotics has vastly improved the therapy of cutaneous bacterial infections, particularly those caused by Staphylococcus aureus. This organism and beta-haemolytic streptococci are the most common cutaneous pathogens. A growing body of evidence suggests that proteins from S. aureus and some strains of streptococci can act as superantigens and cause polyclonal T-cell activation by binding directly to antigen-presenting cells. This process is a likely explanation of Kawasaki's syndrome as well as staphylococcal and streptococcal toxic shock syndrome. Sudden aggravation of atopic dermatitis, contact dermatitis and some cases of psoriasis can be similarly explained. Bacterial toxins can precipitate the staphylococcal scalded skin syndrome. Specific and effective eradication of bacteria and programmes to prevent recurrences are important, particularly in immune suppressed persons. Topical antibiotics used primarily for superficial infections of limited extent and for the prevention of recurrences in carriers of S. aureus should be combined with the use of topical disinfectants. The treatment of selected bacterial skin infections based on clinical examples will be discussed. These include secondarily infected dermatoses, cellulitis and streptococcal carriage in the ano-genital region and staphylococcal folliculitis and nasal carriage.

Costimulatory function of CD28 in avian gammadelta T cells is evolutionarily conserved.

CD28 costimulatory signals are required for T-cell proliferation and lymphokine production. In this work, the functional conservation of CD28 was studied in avian gammadelta T cells. The avian CD28 molecule is expressed on all alphabeta T cells and is capable of giving a costimulatory signal. Most peripheral gammadelta T cells are CD28 negative; however, we identified a CD28-positive gammadelta T-cell subset from peripheral blood comprising about 12% of gammadelta T cells. The peripheral CD28+ gammadelta T-cell subset included all CD8+ gammadelta T cells known to be a responding subset during activation. After polyclonal activation, the frequency of CD28+ gammadelta T cells was increased and the activation also up-regulated CD5, CD25 and major histocompatibility complex (MHC) class II molecules. These changes were detected after both polyclonal and antigen-specific T-cell activation. In addition, we also showed that CD28 can give a costimulatory signal to gammadelta T cells and that this signal leads to up-regulation of IL-2 and bcl-x transcripts. These results indicate that the function of CD28 is evolutionarily conserved and can already be detected in avian gammadelta T cells.

Characterization of lymphocytes in the adult rat testis by flow cytometry: effects of activin and transforming growth factor beta on lymphocyte subsets in vitro.

The rat testis is considered to be an immunologically privileged site because of its reduced capacity to support antigen-specific immune responses. To understand this phenomenon, it is essential to characterize both the lymphocyte subpopulations normally present in the testis and their regulation by testicular cytokines. Peripheral blood was obtained from adult male Dark Agouti or Sprague-Dawley rats, and testicular interstitial tissue was collected after perfusion of the testes to remove blood. Blood and testis lymphocytes were isolated using discontinuous Percoll density gradients, and the testicular lymphocytes were further purified by selective adherence to remove mononuclear phagocytes. The isolated lymphocytes were analyzed by flow cytometry using specific monoclonal antibodies and fluorescein labeling and were enumerated as total T cells, CD4+ T cells, CD8+ T cells, B cells, and natural killer (NK) cells. In contrast to peripheral blood, in which the CD4+ T-cell subset was the major lymphocyte subset, rat testis T cells were predominantly of the CD8+ subset, and a large population of NK cells also were present. Subsequently, peripheral blood lymphocytes were stimulated with the polyclonal T-cell activator, phytohemagglutinin, and cultured in the presence of activin, inhibin, or transforming growth factor beta (TGFbeta) prior to flow cytometric analysis. Activin and TGFbeta suppressed T-cell proliferation without any selective effect on either T-cell subset, and inhibin had no effect. The predominance of CD8+ T cells and NK cells, and the relatively minor proportion of CD4+ T cells, are consistent with both increased cellular immune surveillance and a reduced capacity for initiating antigen-specific immune responses in the adult rat testis.

Immune cell functions in industrial workers after exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin: dissociation of antigen-specific T-cell responses in cultures of diluted whole blood and of isolated peripheral blood mononuclear cells.

A comparative analysis was performed of the phenotype and function of peripheral blood leukocytes of two age-matched cohorts of industrial workers in chemical plants, one of which was exposed occupationally to high concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Median actual TCDD burdens were 116 ng/kg and 4 ng/kg, respectively. The phenotype analysis of peripheral blood mononuclear cells (PBMC) revealed no significant differences in the proportions of CD3, CD4, or CD8+ T lymphocytes, of CD16+ natural killer cells, and of CD19+ B lymphocytes. However, in PBMC of the TCDD-exposed workers; the proportion of CD8+ memory T cells (CD45R0+) was significantly higher, and that of lymphocytes with naive phenotype (CD45RA+) was significantly lower than in PBMC of the control group. Polyclonal and antigen-specific T-cell activation was assessed in parallel in isolated PBMC as well as in diluted whole blood cultures. In both culture systems the polyclonally stimulated cytokine release did not differ significantly between the two cohorts; however, we found a significantly reduced interferon gamma release in diluted whole blood cultures but not in isolated PBMC cultures of the TCDD-exposed cohort when we performed an antigen-specific T-cell stimulation with tetanus-toxoid. Therefore, we propose that exposure of individuals to high doses of TCDD can partially impair in the "blood milieu" those T-cell/monocyte interactions that are essential for antigen-specific T-cell responses, whereas isolated PBMC of the same donors appear functionally less affected.

Consistent transient transfection of DNA into non-transformed human and murine T-lymphocytes

The ability to analyze transcriptional regulation in non-transformed T-cells has been hampered by the inability to reproducibly transiently transfect these cells with DNA constructs. We have previously demonstrated that normal human whole mononuclear and CD4 T-cells can be consistently transiently transfected with plasmid DNA. Human cells were most receptive to plasmid DNA uptake between 19.5 and 20 h after prestimulation with a submitogenic dose of the polyclonal T-cell activator, PHA. Here, we report an alteration and optimization of this protocol for non-transformed murine splenic T-cells, using concanavalin A instead of PHA as the preactivation stimulus. When coupled with the high sensitivity of luciferase reporter gene constructs, this protocol facilitates the analysis of a variety of T-cell-specific promoters in non-transformed T-cells. In addition, we directly demonstrate that murine T-cells are specifically transiently transfected among a population of whole mononuclear cells by using an expression vector for green fluorescent protein.