PHA-stimulated T-lymphocytes Research Articles

Immunotolerant indoleamine-2,3-dioxygenase is increased in condyloma acuminata.

The tryptophan-depleting enzyme indoleamine-2,3-dioxygenase (IDO) is critical for the regulation of immunotolerance and plays an important role in immune-associated skin diseases. To analyse the level of IDO in condyloma acuminata (CA) and its role in this condition. IDO expression was assessed in the skin and peripheral blood of healthy controls and patients with CA. To assess the role of skin IDO in immunity, the ability of isolated epidermal cells to metabolize tryptophan and the influence on polyclonal T-cell mitogen (PHA)-stimulated T-cell proliferation were explored. IDO median fluorescence intensities in peripheral blood mononuclear cells from patients with CA were similar to those from healthy controls. Immunohistochemistry showed that IDO+ cells were rare in normal skin and the control skin of patients with CA, but were greatly accumulated in wart tissue. Most fluorescence signals of IDO+ cells did not overlap with those of CD1a+ Langerhans cells. Human papillomavirus (HPV) DNA probe in situ hybridization showed a large number of IDO+ cells in the HPV- site. Keratinocytes in the skin of healthy controls and the circumcised skin of patients with CA could minimally transform tryptophan into kynurenine, but IDO-competent epidermal cells from warts could transform tryptophan. In addition, these IDO-competent epidermal cells could inhibit PHA-stimulated T-cell proliferation. The addition of an IDO inhibitor, 1-methyl-d-tryptophan, restored the inhibited T-cell proliferation. Abnormally localized high IDO expression might be involved in the formation of a local immunotolerant microenvironment.

Pre-deployment differences in glucocorticoid sensitivity of leukocytes in soldiers developing symptoms of PTSD, depression or fatigue persist after return from military deployment.

Deployed soldiers are at risk of developing stress-related conditions, including posttraumatic stress disorder (PTSD), major depressive disorder (MDD), and severe fatigue. We previously observed condition- and cell-specific differences in sensitivity of immune cells for regulation by glucocorticoids (GCs) pre-deployment between male soldiers with and without subsequent development of high levels of these stress-related symptoms. Here we investigated whether these pre-deployment dysregulations in GC-sensitivity of immune cells persisted after return from military deployment. In a prospective, longitudinal study including 721 male and female soldiers, the in vitro GC-sensitivity of monocytes and T-cells was assessed prior to deployment and one and six months post-deployment. Differences in the longitudinal course of sensitivity for regulation by dexamethasone (DEX) of LPS-stimulated TNF-α production and PHA-stimulated T-cell proliferation between soldiers with and without subsequent symptom development were investigated using linear mixed models. Within the whole group, DEX-sensitivity of monocytes was significantly decreased at six months post-deployment compared to the assessments pre-deployment and one month post-deployment. The DEX-sensitivity of T-cells did not significantly change over time. Participants developing high levels of PTSD symptoms showed high DEX-sensitivity of T-cells, while participants developing high levels of depressive symptoms showed low DEX-sensitivity of T-cells before deployment that persisted at the two time points after return. In addition, participants developing severe fatigue had low DEX-sensitivity of monocytes at all assessments. Our finding that the previously observed pre-deployment group differences in peripheral GC-sensitivity persisted until at least six months after return indicates that in vitro GC-sensitivity of T-cells and monocytes may represent a persistent biological vulnerability factor for development of stress-related conditions PTSD, depression and fatigue.

Only High Concentration of Immunoglobulin Inhibits Activation of Lymphocytes: Potential Use of Immunoglobulin for Preventing Gvhd

AbstractAbstract 4376High-dose intravenous immunoglobulin (IV-IG) is used to treat many autoimmune and inflammatory disorders, yet precise mechanisms have not been understood. In organ transplant, some studies demonstrated that IV-IG therapy, even moderate-dose, given to highly sensitized patients led to reduction in allo-sensitization, fewer acute rejection episodes, and better long-term results for cardiac and renal allograft recipients. Although historically IV-IG has been widely used for mainly prophylaxis of CMV infection in hematopoietic stem cell transplantation, only a few reports mentioned that IV-IG contributed to reduce GVHD severity. The aim of this study is to explore whether IV-IG has immunomoduraroty role against cells involved in GVHD developments or not, if so, appropriate concentration of immunoglobulin and administration schedule for clinical settings should be discussed. First, mixed lymphocyte reaction (MLR) was performed using irradiated dendritic cells (DCs) from C57BL6 as stimulators and splenocytes from Balb/c as responders with immunoglobulin. MLR was strongly inhibited when the concentration was above 25 mg/mL. Next, to identify when immunoglobulin give immunomoduratory function to DCs or T-cells, or both, immunoglobulin treated DCs or splenocytes for 24h before MLR were prepared and washed to remove extra immunoglobulin, then MLR was performed without addition of immunoglobulin. MLR was not suppressed when not added to immunoglobulin during reactions, suggesting that sustained immunoglobulin interaction to DCs, T-cells, or both could be required for sustained immunomoduratory effects. Next, to clarify whether IV-IG inhibit T-cell proliferation via DCs or T-cells directly, we stimulated splenocytes (Balb/c) with phytohemagglutinin (PHA) in the presence of various concentrations of immunoglobulin. Immunoglobulin inhibited PHA-stimulated T-cells, with similarity to the result of MLR using DCs as stimulators as shown in the figure below. It suggested that IV-IG mainly act on T-cells directly, not to DCs. Similar results were obtained using human samples. Finally, to elucidate whether immunoglobulin induce arrest of T-cell proliferation with increased apoptotic cells or not, we measured the early apoptotic cells using annexin V and 7-AAD staining combined with CFSE proliferation assay. Only a few early apoptotic cells were increased when immunoglobulin was added to PHA-stimulated T-cells, demonstrating that IV-IG immunomoduratory effects were mainly brought about T-cell arrest. In conclusion, high concentration of immunoglobulin, in which only high dose IV-IG could be achieved in vivo, contributes to profound and sustained inhibition of T-cell activation. To use IV-IG to prevent GVHD in clinical settings, much higher dose of IV-IG than ever might be needed. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Topoisomerase II levels and G2 radiosensitivity in T-lymphocytes of women presenting with breast cancer

Previous studies from our laboratory have identified a link between intracellular topoisomerase IIα (topo IIα) levels and chromosomal radiosensitivity, as measured by the frequencies of chromatid breaks in the so-called G2-assay. Lower topo IIα levels were associated with reduced chromosomal radiosensitivity in cultured human cells. These findings supported a model, in which it is proposed that such chromatid breaks are the result of radiation-induced errors made by topoisomerase IIα during decatenation of chromatids. Studies from our and other laboratories, using the G2-assay, have shown that phytohaemagglutinin (PHA)-stimulated peripheral blood T-lymphocytes from 40% of female breast cancer cases show elevated chromatid break frequencies when exposed to a small standard dose of ionizing radiation, i.e. elevated above the 90th percentile of a group of female control samples. In the present study we have used a modified G2-assay to test whether elevated frequency of chromatid breaks in breast cancer cases is linked with elevated intracellular topo IIα level in PHA-stimulated T-lymphocytes, and also whether there is a general correlation between chromosomal radiosensitivity and topo IIα level. Our results confirm previous studies that 40% of breast cancer cases show elevated radiosensitivity as compared with controls. Also, the mean chromatid break frequency in breast cancer cases was significantly higher than in controls (P = 0.0001). We found that the mean topo IIα level in the cohort of breast cancer cases studied was significantly raised, as compared with controls (P = 0.0016), which could indicate a genetic propensity towards a raised intracellular production of topo IIα in these individuals. There was no direct correlation between chromosomal radiosensitivity and topo IIα level for individual samples either in the breast cancer cohort or in controls. However, a comparison between control and breast cancer samples shows a higher mean topo IIα level in breast cancer samples that correlates with the elevated mean chromatid break frequency seen in these patient samples. We found no meaningful correlations between either chromatid break frequency or topo IIα level and either tumour grade or hormone status. We conclude that elevated intracellular topo IIα level is likely to be a significant factor in determining the chromosomal response of stimulated T-lymphocytes from certain breast cancer cases.

Negative regulation of IL-17 production by OX40/OX40L interaction

The T-cell cytokine IL-17 is implicated in multiple inflammatory diseases through its induction of several pro-inflammatory cytokines and chemokines in a broad range of cell targets. Production of IL-17 defines the Th17 subset of helper T-cells associated with protection against microorganisms, a profile best characterized in the murine system. Multiple regulators of Th17 cell differentiation and IL-17 production are reported, but the impact of OX40L is not described. OX40 ligand (OX40L) is an early-stage activator of T-cells through its interaction with CD134 (OX40) that is up-regulated on antigen challenged T-cells. Here, we show that OX40L suppresses IL-17 production by PHA-stimulated human PBMC and purified CD4 and CD8 cells. In agreement with prior reports, OX40L signaling through CD134 increased IFNγ and IL-4, both of which are reported to inhibit the production of IL-17. OX40L suppression of IL-17 was completely reversed by a neutralizing IFNγ antibody while there was no effect with a neutralizing IL-4 antibody. Moreover, OX40L also suppressed IL-17 in the presence of IL-23, an established inducer of IL-17 and differentiation factor for Th17 cells. Presuming mediation by IFNγ, we evaluated expression of this cytokine in the presence of OX40L and IL-23. Surprisingly, IL-23 also induced IFNγ by PHA-stimulated T-cells and this effect was enhanced in the presence of OX40L. Addition of the IFNγ antibody not only reversed the OX40L suppression of IL-17 in the presence of IL-23, it markedly enhanced the level of IL-17. These results further establish IFNγ as a primary modulator of IL-17 production in the human cells, much as in the murine system.

An Alternative Transcript of the PANE1 Gene Encodes a Minor Histocompatibility Antigen That Is Selectively Expressed in Resting CD19+ Cells and B-CLL.

We recently identified an HLA-A3-restricted minor histocompatibility antigen (mHAg) encoded by an alternative transcript (transcript k) of the PANE1 gene within an exon unique from all other PANE1 transcripts. Differential CTL recognition of mHAg+ and mHAg− cells is due to a single nucleotide polymorphism that replaces an arginine codon (CGA) with a translation termination codon (TGA). Cytotoxicity assays revealed robust recognition of HLA-A3+ EBV-LCL, minimal recognition of unfractionated peripheral blood mononuclear cells and PHA-stimulated T-cell blasts (PHA-T), and no recognition of dermal fibroblasts. To determine whether the restricted tissue distribution of the mHAg correlated with transcript k expression, we developed a real-time quantitative PCR assay to specifically detect transcript k. In normal tissues, expression was highest in spleen, low in all other tissues examined, and undetectable in dermal fibroblasts. Analysis of resting and activated peripheral blood cell fractions revealed very high expression levels in resting CD19+ cells, intermediate levels in EBV-LCL and resting CD4+ and CD8+ cells, and very low levels in activated CD19+ cells as well as resting and activated mononuclear cells, CD14+ cells, and activated CD4+ and CD8+ cells. EBV-LCL from mHAg− TGA homozygotes express significantly lower transcript k levels (p=0.043; Student's t test) than mHAg+ EBV-LCL, suggesting the possibility of nonsense-mediated decay. The preferential expression of transcript k in normal resting CD19+ cells prompted us to investigate expression in malignant CD19+ cells. In a small sample of CD19+ acute lymphoblastic leukemia cells, we observed low-level expression comparable to that of activated normal CD19+ cells. In contrast, we observed high-level expression in CD19+ B-CLL cells comparable to or greater than that of normal resting CD19+ cells, and far greater than the levels in any other cell type tested. CTL cytotoxicity assays of a subset of HLA-A3+, mHAg+ B-CLLs revealed that they uniformly presented the mHAg encoded by PANE1 transcript k. Our finding that transcript k was selectively expressed in resting CD19+ cells and CD19+ B-CLL contradicted a previous report (Bierie et al., Gene Expression Patterns 4:389, 2004) that the longest PANE1 transcript (transcript c) is preferentially expressed in activated lymphoid cells. We therefore evaluated transcript c expression in the same panel of samples, and confirmed that it was selectively expressed in activated CD19+ cells, with very low levels in resting CD19+ and primary B-CLL cells. Thus, PANE1 transcripts k and c display reciprocal expression in resting and activated CD19+ cells and primary CD19+ B-CLL cells. To determine if activation of CD19+ cells would increase expression of transcript c and decrease expression of transcript k, we evaluated the effect of CD40L stimulation in 6 primary B-CLL samples. In all samples tested, CD40L stimulation led to a profound decrease in transcript k expression, and a significant increase in transcript c expression. These studies suggest distinct roles for different PANE1 isoforms in resting versus activated CD19+ cells, and identify PANE1 as a potential therapeutic target in B-CLL.

RNA Electroporation as a New Gene Transfer Method in Hematopoietic Progenitor Cells, Mesenchymal Cells and Activated T-Cells.

Electroporation of messenger RNA has become an established method for gene transfer into dendritic cells for immunotherapeutic purposes. When electroporating dendritic cells, this method combines high transient gene expression with low cell mortality without the risk for insertional mutagenesis. Here, we have investigated the potential of mRNA electroporation to induce short-term transgene expression in other cell types such as bone marrow CD34+ progenitor cells, in vitro cultured bone marrow mesenchymal cells and phytohemagglutinin A (PHA)-stimulated peripheral blood T-cells. Transgene expression after electroporation with mRNA encoding the enhanced green fluorescent protein (EGFP) was evaluated by flow cytometry. Flow cytometric analysis 24h after EGFP mRNA electroporation revealed that 35% of fresh uncultured bone marrow-derived CD34+ hematopoietic progenitor cells was efficiently transfected. In the population of in vitro cultured mesenchymal cells, 90% of the viable CD45-CD13+ cells showed detectable EGFP expression. The level of transient EGFP expression in the PHA-stimulated T-cell population was 47% at 24h after electroporation. Importantly, no significant difference of cell mortality between electroporated and non-electroporated cells was observed, due to the mild electroporation procedure ensuring high cell viability. When looking at the kinetics of EGFP expression, we saw that EGFP was still expressed at day 7 after electroporation with highest expression at day 4. Short-term gene introduction by mRNA electroporation in hematopoietic progenitor cells, mesenchymal cells and T-cells could be used to direct the differentiation and/or to modulate the function of these cells. We conclude that mRNA electroporation is an efficient method for short-term gene transfer in CD34+ cells, mesenchymal cells and activated T-cells with potential applications in gene therapy procedures.

Transient CD4/CD8 ratio inversion and aberrant immune activation during dengue virus infection.

The immune status after dengue virus infection was studied in dengue patients from an outbreak of serotype 3 dengue virus infection in the southern part of Taiwan during November and December 1998. Consecutive blood samples from 29 dengue patients, of whom 21 had dengue fever and 8 had dengue hemorrhagic fever/dengue shock syndrome, were collected, and the immunophenotypes of the peripheral blood mononuclear cells were determined by flow cytometry. The early activation marker CD69 appeared on lymphocytes and monocytes at day 4 after the onset of fever, and declined afterward. However, a transient reverse in the CD4/CD8 ratio occurred at days 6-10 after the onset of fever. The CD4/CD8 ratio inversion was manifested in 10 of 29 dengue patients and was encountered more frequently in dengue hemorrhagic fever/dengue shock syndrome than in dengue fever patients. Analysis of the clinical blood cell count of these 10 cases showed that increase of immature neutrophils developed at fever days 5-6, CD4(dim) or CD8(dim) monocytosis at days 6-7, and atypical lymphocytosis at days 8-10 after the onset of fever. Serum IL-6 was found at either day 7 or day 9-11. The PHA-stimulated T-cell response was depressed as well. These changes in immune parameters indicate aberrant immune activation during dengue virus infection and might be involved in the pathogenesis of dengue virus infection.

Effects of imipenem and cilastatin on human T-lymphocytes derived from acute leukemia patients with chemotherapy-induced leucopenia: studies of T-lymphocyte responses in the presence of acute myelogenous leukemia (AML) blast accessory cells

The effects of imipenem and cilastatin on human T-lymphocytes were studied in vitro. As responder T-cells were used T-lymphocyte clones derived from acute leukemia patients with chemotherapy-induced cytopenia, and the accessory cells were highly enriched acute myelogenous leukemia (AML) blasts. The effects of imipenem and cilastatin on phytohemagglutinin (PHA), anti-CD3 and anti-CD3+anti-CD28 stimulated activation were assayed, and in addition drug effects on cytokine-dependent proliferation of activated T-lymphocytes were investigated. Imipenem inhibited IL2-dependent proliferation of activated CD4 + and CD8 + T-cell clones, and an inhibition was also detected for IL7-, IL12-, IL15-, IL16- and IL17-dependent clonal proliferation. Imipenem caused a weak inhibition of anti-CD3- and PHA-stimulated T-cell proliferation when using 50 Gy irradiated AML blast accessory cells derived from various patients, whereas no effect was observed for anti-CD3+anti-CD28 stimulated and allostimulated activation. Imipenem decreased the release of IL4 and Interferon-γ by T-cell clones stimulated with anti-CD3 and PHA in the presence of native (nonirradiated) AML blasts. The imipenem effects were observed at concentrations corresponding to levels reached in vivo, whereas even high concentrations of cilastatin did not alter T-cell responses. The T-lymphocyte inhibition is probably caused by a direct effect of imipenem on the T-cells.

Co-cultivation of CD4 + and CD8 + human T-cells leads to the appearance of CD4 cells expressing CD8 through de novo synthesis of the CD8 α-subunit

The appearance of a population of dual-staining CD4 +CD8 + cells in human T-lymphocyte cultures has been reported by various authors, including our own observation that they are always seen in simple phytohaemagglutinin-stimulated cultures from several different donors. The purpose of the present study was to investigate factors involved in the dual-staining (DS) phenotype, and to clarify some apparent inconsistencies between published observations. Our findings can be summarised as follows. 1. A population of DS CD4 +CD8 + cells always appears in PHA-stimulated T-cell cultures if they contain both CD4 and CD8 subsets. The incidence of DS cells is related to PHA concentration, but other factors are involved since DS cells are not seen in PHA-stimulated cultures of purified CD4 + or CD8 + cells. Stimulation with PHA is not a prerequisite since very similar results are seen with ConA. 2. Direct physical contact between CD4 + and CD8 + cells is required for the appearance of the DS phenotype; soluble factors alone, including IL-4, appear not to be responsible. 3. The DS phenotype in these conditions is always CD4 + cells weakly expressing CD8 and is a consequence of de novo synthesis of the CD8α molecule by the CD4 + cells.

Identification of lymphocyte 5-HT 3 receptor subtype and its implication in fish T-cell proliferation

In the present study, we identified the serotonergic receptor of type 3 (5-HT 3) on the lymphocytes of a teleost fish, Oncorhynchus mykiss. In the pharmacological studies on the binding of [ 3H]serotonin to membrane receptor sites, 2-methyl-5-HT, an agonist of 5-HT 3 receptors, displaced the binding of [ 3H]serotonin to fish lymphocytes, indicating the presence of 5-HT 3 receptors on these cells. The known antagonists of the mammalian 5-HT 3 receptor, ICS-205-930 and metoclopramide, failed to displace [ 3H]serotonin binding to lymphocytes during the period of association equilibrium (8 min); however, these antagonists progressively displaced [ 3H]serotonin binding from 10 to 40 min of incubation. These results suggest that fish 5-HT 3 lymphocyte receptors may differ pharmacologically from mammalian receptors. As mammalian 5-HT 3 receptors are coupled with Na + inward movements, we undertook a study on Na + influx by using SBFI AM , a fluorescent probe. In SBFI AM loaded fish lymphoc 2-methyl-5-HT leaked Na + inward movements. Prior incubation of lymphocytes for 30 min in the presence of 5-HT 3 antagonists, ICS-205-930, metoclopramide and MDL-72222, curtailed significantly the Na + influx evoked by 2-methyl-5-HT, demonstrating that Na + is leaked into fish lymphocytes via the 5-HT 3 receptor-channel whose functioning is blocked by these antagonists. Furthermore, 2-methyl-5-HT exerted immunosuppressive effects in a dose dependent manner on fish T-lymphocytes stimulated by phytohaemagglutinin (PHA). Serotonin and 2-methyl-5-HT blocked the cell cycle progression of PHA-stimulated T-cells from G 0 G 1 to S phase. The immunosuppressive effects of 2-methyl-5-HT on T-cells were partially reversed by the antagonists, metoclopramide and ICS-205-930; however, the latter antagonist at high concentrations synergized with the immunosuppressive effects of 2-methyl-5-HT. These results demonstrate that the fish lymphocyte 5-HT 3 receptor, which may be pharmacologically different from mammalian receptor subtype, is functionally implicated in fish T-cell proliferation.

Defective interferon-gamma production by T-lymphocytes from patients with acute brucellosis.

The authors investigated the production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) by phytohaemagglutinin (PHA)-stimulated T-lymphocytes from 21 untreated patients with acute brucellosis. PHA-stimulated T-lymphocytes from acute brucellosis patients showed normal IL-2 production but defective IFN-gamma production (brucellosis patients 531 +/- 103 pg mL-1 vs. healthy controls 1024 +/- 212 pg mL-1) after 72 h of culture. This altered pattern of IL-2 and IFN-gamma production by T-lymphocytes was observed in seven brucellosis patients whose T-lymphocytes exhibited a normal proliferative response to PHA (61 612 +/- 18 422 cpm) as well as in the 14 patients with a defective T-lymphocyte proliferative response to the PHA after 5 days of culture (19 479 +/- 4409 cpm). After antibiotic therapy, production of the two lymphokines by the PHA-stimulated T-lymphocytes from acute brucellosis patients was similar to that of T-lymphocytes from healthy control subjects. The authors conclude that PHA-stimulated T-lymphocytes from untreated patients with acute brucellosis have defective INF-gamma production but normal IL-2 production.

Inhibition of re-expression of surface CD4, but not CD8, on activated human T-lymphocytes by the immunosuppressive drugs dexamethasone and cyclosporine a: Correlation with inhibition of proliferation

Our objective was to study the re-expression of CD4 and CD8 on activated human T-lymphocytes in the presence of dexamethasone and cyclosporine A at physiologically relevant concentrations. Peripheral blood mononuclear cells from 12 healthy volunteers were cultured for up to 72 h in the presence and absence of phytohaemagglutinin A (PHA) and dexamethasone and cyclosporine A at a range of concentrations. Expression of surface CD3, CD4 and CD8 and the activation marker CD25 was quantified on T-lymphocytes using flow cytometry. T-lymphocytes cultured with PHA showed a significant ( P<0.01) reduction in the expression of surface CD3, CD4 and CD8 compared with cells cultured in medium alone, which was apparent as early as 0.5 h after PHA exposure. The reduction in the expression of both CD4 and CD8 in the first 0.5 h after exposure correlated with that of CD3 ( P<0.02). Expression of CD4 and CD8 subsequently increased again, so that by 48 h after exposure CD4 expression was no longer significantly different to that of cells cultured in medium alone. CD8 expression recovered later, but within 72 h. Both dexamethasone and cyclosporine A, when added at the commencement of the cultures, significantly inhibited, in a concentration-dependent (10 −6-10 −9mol/1) fashion, the re-expression of CD4, but not CD8, on the PHA-stimulated T-lymphocytes measured after 48 h. In contrast, CD25 expression on both CD4 and CD8 T-lymphocytes was inhibited by both drugs to an equivalent extent over the same range of concentrations. At this time point, the degree of inhibition of CD4 re-expression by both drugs correlated significantly with the degree of inhibition of CD25 expression. We conclude that activation of T-lymphocytes results in rapid down-modulation of surface CD3, CD4 and CD8 followed by re-expression which is inhibited in the case of CD4, but not CD8, by dexamethasone and cyclosporine A. Since CD4 is thought to co-operate in T-lymphocyte antigen recognition by the CD3-antigen receptor complex, this effect may contribute to the immunosuppressive effect of these drugs on CD4 T-lymphocytes.

The essentiality of folate for the maintenance of deoxynucleotide precursor pools, DNA synthesis, and cell cycle progression in PHA-stimulated lymphocytes.

The fidelity and progression of DNA synthesis is critically dependent on the correct balance and availability of the deoxynucleoside triphosphate (dNTP) precursors for the polymerases involved in DNA replication and repair. Because folate-derived one-carbon groups are essential for the de novo synthesis of both purines and pyrimidines, the purpose of this study was to determine the effect of folate deprivation on deoxynucleotide pool levels and cell cycle progression. Primary cultures of phytohemagglutin (PHA)-stimulated splenocytes were used as the cellular model. T-cells and macrophages were purified from spleen cell suspensions obtained from F344 rats and recombined in culture. The cells were harvested after a 66-hr incubation with PHA and analyzed for nucleotide levels by reverse-phase HPLC with diode array detection. The proportion of cells in the different phases of the cell cycle was determined by bivariate flow cytometric measurement of bromodeoxyuridine (BrdU) incorporation and DNA content (propidium iodide staining). PHA-stimulated T-cells cultured in medium lacking folate and methionine manifested significant decreases in the deoxynucleotides dCTP, dTMP, dGTP, and dATP relative to cells cultured in complete medium. The reduction in dNTP pools was associated with a decrease in the corresponding ribonucleotide pools. Flow cytometric analysis revealed a 2-fold increase in S and G2/mitosis (G2/M) DNA content in PHA-stimulated cells cultured in the medium lacking folate and methionine, which suggests a delay in cell cycle progression. These alterations in DNA content were accompanied by a 5-fold decrease in BrdU incorporation relative to PHA-stimulated cells cultured in complete medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin and IGF-I stimulate normal and virally transformed T-lymphocyte cell growth in vitro

We used normal and HTLV-II-transformed T-lymphocytes as target cells to study clonal proliferative responses to physiologic and supraphysiologic concentrations of insulin and IGF-I. Responses of both growth factors were measured in the presence and absence of αIR-3, an IGF-I receptor-blocking antibody. A biphasic response to insulin was noted in all cell lines with the first peak [78 ± 6.6% (means ± SE) above control] occurring at 1.4 or 1.6 nmol/liter and a second peak (84 ± 4.9% above control) occurring at 18.0 nmol/liter. Following preincubation with αIR-3, the overall clonal profile in response to insulin was significantly reduced [ F(7,56) = (10.4, p < .0001] as a result of blunting at high physiologic and supraphysiologic insulin concentrations, i.e., ≥1.6 nmol/liter. As expected, the overall clonal profile in response to IGF-I was blocked by αIR-3 [ F(4,32) = 11.6, p < .0001]. These data show that insulin at both physiologic and supraphysiologic concentrations, as well as IGF-I, stimulate virally transformed T-lymphoblast growth. The significant inhibition of growth responses to high concentrations of insulin and to IGF-I by αIR-3 suggests mediation of these effects through the IGF-I receptor. Similar studies were performed using freshly isolated, phytohemagglutinin (PHA)-stimulated T-lymphocytes. The overall response to insulin was significantly reduced compared to the profile of transformed T-lymphoblasts [ F(7,70) = 4.9, p = .0002] as a result of blunting at physiologic insulin concentrations <1,8 nmol/liter. In response to IGF-I, the clonal profile of PHA-stimulated T-lymphocytes was slightly reduced compared to that of virally transformed T-lymphoblasts [ F(4,40) = 3.4, p = .0174]. Thus, both insulin and IGF-I receptor-effector mechanisms are involved in the growth of virally transformed T-lymphoblasts, whereas the IGF-I receptor-effector mechanism appears to play a more significant role in the growth of normal, mitogen-activated T-lymphocytes.

Valid estimation of IL2 secretion by PHA-stimulated T-cell clones absolutely requires the use of anti-CD25 monoclonal antibody to prevent IL2 consumption

A major problem encountered for quantification of IL2 production by stimulated T cells is its simultaneous consumption by these activated cells. In the present study, 40 T-cell clones (TCC) derived from normal peripheral blood, hyperplastic lymph nodes (LN) or lymph nodes involved by malignant lymphomas, were studied for their ability to produce IL2. When supernatants were generated in the presence of 20% fetal calf serum (FCS), no IL2 could be detected for 22 of the 40 TCC, whereas very low levels were found for the 18 other TCC (mean value 31 pg/ml; range from 10 pg/ml to 114 pg/ml); in contrast, when conditioned media were produced with reduced amounts of FCS (final concentration, 1%) as well as in the presence of an anti-CD25 monoclonal antibody (final concentration, 50 μg/ml), all TCC were found to release IL2, and very high quantities of this lymphokine were measured (mean value: 11 387 pg/ml; range, from 250 pg/ml to 37 000 pg/ml). Consequently, inhibition of IL2 consumption by PHA-stimulated TCC seems to be an absolute requirement for estimating the true capacity of T cells to produce this lymphokine.

The additive effect of certain drugs on the Cyclosporine A inhibition of human T‐cell proliferation

The inhibitory effect of Cyclosporine A (CsA) when combined with theophyllamine, warfarin, verapamil or dipyridamol on in vitro proliferation of human T lymphocyte was investigated. All drugs caused an additional inhibition of PHA activation compared with CsA alone, also when the CD8-negative and -positive subsets were tested separately. This effect was dose-dependent for all drugs. Theophyllamine, verapamil and warfarin had to be added early during PHA-stimulated T-cell activation to cause maximal inhibition. Also when testing the proliferative response in mixed lymphocyte cultures, the drugs showed a dose-dependent additional inhibition compared with CsA alone. CsA alone caused no inhibition of lymphokine-dependent growth of T-cell lines, whereas all four drugs caused a weak inhibition in this test system.

Synthesis of the third component of complement (C3) by lectin-activated and HTLV-infected human T-cells

The third component of complement (C3) plays key roles in complement activation of both the classical and alternative pathways. The liver is the major site of C3 synthesis; monocytes, B-lymphocytes and leukemic cell lines of the myeloid lineage also synthesize C3. Here we report that the C3 gene is inactive in fresh T-cells, but active in T-cells treated with the lectin phytohemagglutinin (PHA). Northern blot hybridization studies show that PHA-activated T-cells and all the T-cell lines tested express the 5.3 kb RNA transcript reported for C3 in HepG2, a hepatoma cell line, and monocytes. We used radioimmune precipitation followed by polyacrylamide gel electrophoresis to show that PHA-stimulated T-cells and T-cell lines, which are not infected with the human T-lymphotropic virus (HTLV), synthesize and release C3 proteins with molecular masses of 185, 115 and 80 kD; HTLV-infected T-cell lines release C3 proteins of 170, 115 and 70 kD. In contrast, monocytes produced C3 proteins of 115 and 70 kD similar to the serum form of this protein. The role of T-lymphocyte C3 and the implications of HTLV-infection are discussed.

Effect of histamine on the T-cell colony formation of PHA-stimulated cells

The effect of histamine on T-cell colony formation was studied in human peripheral blood mononuclear cells. Histamine inhibited dose-dependently (10(-4)-10(-6) M) the colony formation of PHA-stimulated T-cells. The inhibition was similar in normal controls and rheumatoid arthritis (RA) patients in spite of the fact that in RA the colony formation was significantly lower than in the normal controls. No increase of colony formation was observed at low concentrations (less than 10(-7) M). Impromidine was less effective than histamine, and pyridylethylamine (PEA) was inactive. Cimetidine counteracted the effect of histamine while chlorpheniramine did not. The results show that colony formation may be inhibited through H2-receptors. This action may be of importance in cellular interactions in tissues with high local histamine concentrations.

Murine polyclonal T-lymphocyte activation induced by phytohemmagglutinin; Differential lymphokine requirements of two unusual activation pathways defined by resistance to blockade by barium and by cyclosporin A

We have recently demonstrated that polyclonal T-cell activation induced by PHA defines an activation pathway which is resistant to blockade by barium (Ba 2+) ions. Other modes of T-cell activation, including ConA-induced responses, are completely blocked by Ba 2+, which seems to affect an early Ca 2+-dependent step of T-cell activation, as determined by kinetic and competition experiments. In the present study, we have analysed the lymphokine requirements of Ba 2+-resistant pathway of PHA-induced T-cell activation by means of functional blocking experiments with monoclonal antibodies (mAbs) directed against mouse IL-2 (mAb S4B6) and against mouse IL-4 (mAb 11B11). We found that Ba 2+-resistant T-cell activation can be blocked by either S4B6 or 11B11. Thus, both IL-2 and IL-4 participate in Ba 2+-resistant T-cell growth induced by PHA. In addition, we found that cyclosporin A (CsA) completely blocks T-cell activation induced by either ConA or by PHA plus Ba 2+, but not T-cell activation induced by PHA in the absence of Ba 2+, which is reduced by less than 50% in most experiments. This CsA-resistant proliferative component of the PHA response is, thus, distinct from the Ba 2+-resistant response, and is carried out by proliferating T-cells. Although mAbs S4B6 and 11B11 are potent blockers of ConA-induced responses, they failed to block CsA-resistant T-cell growth induced by PHA. At the doses of CsA employed, no IL-2 and/or IL-4 activity could be detected in the supernatants of CsA-treated, PHA-stimulated T-cell cultures. The data indicate that this CsA-resistant pathway is both IL-2 and IL-4-independent. The lymphokine involved in this T-cell activation pathway remains to be identified.