Human Peripheral Blood T-lymphocytes Research Articles

Methyl inosine monophosphate (MIMP) augments T-lymphocyte mitogen responses and reverses various immunosuppressants

Methyl inosine monophosphate (MIMP) augments preferentially the in vitro responses of human and murine lymphocytes to a T-cell mitogen such as phytohemagglutinin (PHA) and inconsistently to a B-cell mitogen such as pokeweed or lipopolysaccharide (LPS). In a normal interleukin-2-dependent cell line (CTLL), MIMP showed little or no effect on IL-2 action; however, in a murine CTLL line exhibiting impaired responses to IL-2, MIMP stimulated thymidine incorporation and restored the response to IL-2. MIMP augments the PHA responses of both CD4 + and CD8 + human peripheral blood T-cells. The effect of MIMP to augment the PHA response of human lymphocytes is paralleled by the parent molecule, IMP. MIMP, but not IMP, is resistant to hydrolysis by 5'nucleotidase; thus, MIMP appears to be a protected analogue of IMP which is capable of in vivo action. MIMP (100 μg/ml) augments the PHA responses of 15 of 24 elderly humans. MIMP also augments the PHA responses of eight HIV-infected pre-AIDS patients but not of eight AIDS patients. When PHA responses of human lymphocytes are suppressed in vitro by an HIV-derived immunosuppressive peptide, interferon α, or prostaglandin PGE 2, MIMP (0.1–100 μg/ml) progressively restores the depressed response; however, when the suppression is severe (greater than 50%), MIMP cannot restore the response. These data indicate that MIMP potentiates normal T-lymphocyte mitogen responses and restores those impaired by a variety of inflammatory and immunosuppressive influences.

Radiopaque agents effect on in vitro human peripheral blood T-lymphocyte blast transformation

Radiopaque agents effect on in vitro human peripheral blood T-lymphocyte blast transformation

Influence of PHA stimulation on the cytotoxicity and mutagenicity of X-rays and ethylnitrosourea in human peripheral blood T-lymphocytes

We investigated the effects of mitogenic stimulation on the cytotoxicity and mutagenicity of X-rays and ethylnitrosourea (ENU) in human peripheral blood lymphocytes using a cloning technique. Resistance to 6-thioguanine (TG) served as the genetic marker. Day 10 (unstimulated) lymphocytes were about two times more radiosensitive than day 3 (stimulated) lymphocytes to the cytotoxicity when compared for the D 0 value (0.72 Gy vs. 1.54 Gy), and about five times more radiosensitive to its mutagenicity when compared for the frequency of TG-resistant cells following exposure to 4 Gy of X-rays (2.5 × 10 −6 vs. 126.0 × 10 −6). On the other hand, day 3 (stimulated) lymphocytes were about three times more sensitive to ENU with a D 37 value of 1.03 mM compared with 2.82 mM for day 0 (unstimulated) lymphocytes, but as sensitive as day o lymphocytes to its mutagenic effect. These results indicate that the sensitivity of lymphocytes for cytotoxicity and mutagenicity is modified by mitogen stimulation, when lymphocytes are exposed to carcinogens or mutagens in vitro.

Evidence That TGF-β Can Inhibit Human T-Lymphocyte Proliferation through Paracrine and Autocrine Mechanisms

Transforming growth factor-β (TGF-β) has been documented as having an inhibitory effect on the proliferation and growth of human T-lymphocytes. We examined the relative contribution of both exogenous and endogenous TGF-β to this inhibitory action. Purified human peripheral blood T-cells were cultured with Con A (0.2 μg/ml), washed with methyl mannopyranoside, and then cultured in rIL-2 (5 U/ml) with or without TGF-β (80 p M). Proliferation, as measured by uptake of tritiated thymidine at 72 hr, was inhibited by added active TGF-β. Addition of neutralizing anti-TGF-β antibodies at the initiation of culture abrogated the antiproliferative effects of TGF-β. A mink lung cell bioassay was used to measure endogenous TGF-β production by the T-cells following transient acidification of the supernatants to activate latent TGF-β. T-lymphocytes cultured with rIL-2 alone produced low levels of TGF-β, first detectable at 72 hr. The addition of (active) TGF-β to these cultures resulted in earlier and higher levels of endogenously produced latent TGF-β protein. This was reflected at the mRNA level as well. The exogenously added active TGF-β appeared to be depleted during the culture period, presumably by the activated T-cells, which exhibited elevated levels of types I, II, and Ill TGF-β receptors. The increase in TGF-β protein levels was due to endogenous TGF-β synthesis and secretion as supported by a capture assay using 35 S-labeled culture supernatants. These findings indicate that both paracrine and autocrine mechanisms are involved in the inhibitory effects of TGF-β on the proliferation of normal human T-lymphocytes and suggest that other TGF-β-producing cells can augment production of TGF-β by activated T-lymphocytes.

An improved procedure for the in vitro expansion of human T-lymphocyte clones for mutant analysis

Assays detecting mutants present in human peripheral blood T-lymphocytes have been developed to monitor the population for somatic mutations. In order to investigate the nature of the mutations, colonies are further expanded in vitro by repeated lectin stimulation. To characterise fully each mutant clone, sufficient cells (∼10 7) must be available for several molecular and biochemical techniques to be employed. These techniques, and their importance to the assay for population monitoring, are discussed briefly. We report here that the expansion of mutant colonies to ∼10 7 cells by repeated lectin stimulation is not effective for all T-cell clones but that an alternative “lectin free” expansion method has enabled us to expand all the clones tested from a variety of normal donors and other individuals of interest.

Chemical Nature and Immunotoxicological Properties of Arachidonic Acid Degradation Products Formed by Exposure to Ozone

Ozone (O3) exposure in vivo has been reported to degrade arachidonic acid (AA) in the lungs of rodents. The O3-degraded AA products may play a role in the responses to this toxicant. To study the chemical nature and biological activity of O3-exposed AA, we exposed AA in a cell-free, aqueous environment to air, 0.1 ppm O3, or 1.0 ppm O3 for 30-120 min. AA exposed to air was not degraded. All O3 exposures degraded > 98% of the AA to more polar products, which were predominantly aldehydic substances (as determined by reactivity with 2,4-dinitrophenylhydrazine and subsequent separation by HPLC) and hydrogen peroxide. The type and amount of aldehydic substances formed depended on the O3 concentration and exposure duration. A human bronchial epithelial cell line (BEAS-2B, S6 subclone) exposed in vitro to either 0.1 ppm or 1.0 ppm O3 for 1 hr produced AA-derived aldehydic substances, some of which eluted with similar retention times as the aldehydic substances derived from O3 degradation of AA in the cell-free system. In vitro, O3-degraded AA induced an increase in human peripheral blood polymorphonuclear leukocyte (PMN) polarization, decreased human peripheral blood T-lymphocyte proliferation in response to mitogens, and decreased human peripheral blood natural killer cell lysis of K562 target cells. The aldehydic substances, but not hydrogen peroxide, appeared to be the principal active agents responsible for the observed effects. O3-degraded AA may play a role in the PMN influx into lungs and in decreased T-lymphocyte mitogenesis and natural killer cell activity observed in humans and rodents exposed to O3.

Chemical nature and immunotoxicological properties of arachidonic acid degradation products formed by exposure to ozone.

Ozone (O3) exposure in vivo has been reported to degrade arachidonic acid (AA) in the lungs of rodents. The O3-degraded AA products may play a role in the responses to this toxicant. To study the chemical nature and biological activity of O3-exposed AA, we exposed AA in a cell-free, aqueous environment to air, 0.1 ppm O3, or 1.0 ppm O3 for 30-120 min. AA exposed to air was not degraded. All O3 exposures degraded > 98% of the AA to more polar products, which were predominantly aldehydic substances (as determined by reactivity with 2,4-dinitrophenylhydrazine and subsequent separation by HPLC) and hydrogen peroxide. The type and amount of aldehydic substances formed depended on the O3 concentration and exposure duration. A human bronchial epithelial cell line (BEAS-2B, S6 subclone) exposed in vitro to either 0.1 ppm or 1.0 ppm O3 for 1 hr produced AA-derived aldehydic substances, some of which eluted with similar retention times as the aldehydic substances derived from O3 degradation of AA in the cell-free system. In vitro, O3-degraded AA induced an increase in human peripheral blood polymorphonuclear leukocyte (PMN) polarization, decreased human peripheral blood T-lymphocyte proliferation in response to mitogens, and decreased human peripheral blood natural killer cell lysis of K562 target cells. The aldehydic substances, but not hydrogen peroxide, appeared to be the principal active agents responsible for the observed effects. O3-degraded AA may play a role in the PMN influx into lungs and in decreased T-lymphocyte mitogenesis and natural killer cell activity observed in humans and rodents exposed to O3.

Production and regulation of gelatinase B by human T-cells

Here we describe for the first time the production of gelatinase B (MMP 9; 92-kDa type IV collagenase) by human peripheral blood T-lymphocytes. Expression of this enzyme was found to be dependent on the activation status of these T-cells and to be regulated by Interleukin-2.

Effects of Benzo[ a]pyrene on Concanavalin A-Stimulated Human Peripheral Blood Mononuclear cells in Vitro: Inhibition of Proliferation but No Effect on Parameters Related to the G 1 Phase of the Cell Cycle

The immunotoxic effects of the environmental carcinogen benzo[ a]pyrene (BaP) have been evaluated using mouse, but not human, lymphocytes. Since susceptibility to immunomodulation by BaP may be species-related, the effects of BaP on human peripheral blood mononuclear cells (HPBMC) from several individuals were investigated in vitro. HPBMC were stimulated by the T-cell mitogen concanavalin A (con A) in the presence of BaP (10 −9 to 10 −6 M) or vehicle control (acetone) and evaluated after 3 days for [ 3H]TdR incorporation, cell numbers, cell-cycle distribution, viability, and expression of cell surface or intracellular activation antigens. IL-2 production was quantified at 24 hr. Incorporation of [ 3H]TdR was the parameter most sensitive to BaP with an IC 50 relative to controls of 4.5 ± 3.1 × 10 −8 M with a range for individuals of 1.8 × 10 −8 to 6.6 × l0 −8 M ( n = 15). The P450 inhibitor 7,8-benzoflavone (10 −6 M) partially prevented BaP inhibition of [ 3H]TdR incorporation. Maximum suppression of this parameter required that BaP be present in culture >24 hr. The number of cells recovered from culture was decreased by l0 −8 to 10 −6 M BaP, but viability was only slightly diminished by 10 −7 to 10 −6 M BaP. BaP had little or no effect on the percentages of cells with induced IL-2 (CD25) or transferrin (CD7 I) receptors or on the amount of IL-2 activity present in 24-hr culture supernatants. Despite the BaP-mediated decrease in [ 3H]TdR incorporation, cell-cycle analysis indicated that BaP at 10 −7 and 10 −6 M increased the percentages of cells in S phase, and correspondingly decreased the percentages of cells in the G 0G 1, phase. Also, BaP (10 −7 to 10 −6 M) decreased the percentages of G 0/G 1 cells that were positive for the intracellular activation antigen PCNA (or Ki-67), but had no affect on this percentage if cells were prevented from traversing S phase by mimosine or aphidicolin. These results suggest that BaP inhibits DNA synthesis and proliferation of con A-stimulated, human peripheral blood T-cells at culture concentrations as low as 10 −8 M by a P450-dependent process, but has little or no effect on measured G1-associated events at culture concentrations up to 10 −6 M.

Reassessment of human peripheral T-lymphocyte lifespan deduced from cytogenetic and cytotoxic effects of radiation.

Estimates of the mean lifetime of human peripheral blood T-lymphocytes (PBTL) have ranged from 1.5 to 10 year. To derive an improved estimate, kinetic and dose parameters of four deterministic, two-compartment models of loss and replacement of human peripheral blood lymphocytes (PBL) were fitted to a previously published extensive data set on loss of dicentrics plus rings (DR) in PBL sampled from X-ray-treated ankylosing spondylitis patients for > 20-year-post-treatment, involving a fixed external-dose regimen (with dose to PBL unknown). The four models investigated all incorporated fixed dose-response submodels for radiation-induced cytogenetic damage and cell killing that were based entirely on in vitro data for X-ray-exposed human PBTL. Two of the models are shown to provide a reasonably good prediction not only of the data on DR loss to which the models were fit, but also of corresponding data on recovery of the mean of measured total PBL. The modelling results indicate that it is unlikely that long-lived human recirculating 'memory' PBTL are homogeneous with respect to lifespan, but rather that two major subpopulations, T1 and T2, are involved having mean lifespans (and approximately 95% confidence limits) estimated to be approximately 1.1 (0.76-1.9) and approximately 6.3 (5.8-6.9) year, respectively. One of the two models consistent with the data posits that T1 (possibly 'short-term antigen-memory') cells are approximately 60% of recirculating T-lymphocytes that convert at a rate of approximately 9% year-1 into T2 (possibly 'long-term antigen-memory') cells, and that the T1 cells are virtually all derived from a central-body source such as thymus and/or central lymphatic tissues.

Effects of strong magnetic fields on cell growth and radiation response of human T-lymphocytes in culture.

Experiments were undertaken in order to verify whether or not a strong magnetic field would have any biological effects on the cell growth, viability and radiation response of mammalian cells. Magnetic field exposures were conducted using a superconducting magnet with freshly-isolated human peripheral blood T-lymphocytes maintained at their normal growing temperature of 37 degrees C. The static magnetic fields with intensities up to 6.3-tesla (T) exerted little influence on the cell growth and viability of actively-growing T-lymphocytes under normal cell-culture conditions. On the other hand, the T cells exposed to the magnetic fields (4 T-6.3 T) during PHA stimulation were inhibited in their cell growth when compared to controls. The effects of the magnetic fields with intensities up to 2 T on cell growth properties, however, were minimal in this system. Also, the radiosensitivity of T-lymphocytes previously exposed to the strong magnetic fields was more sensitive than that of control cells. These results suggest that exposure to a static magnetic field of 4 T or stronger might lead to physiological and growth abnormalities at the cellular level.

Use of a murine T-cell hybridoma expressing human T-cell receptor α- and β-gene products as a tool for the production of human T-cell receptor-specific monoclonal antibodies

We describe the production of mouse monoclonal antibodies specific for the human TcR using as the immunogen transfected murine T-cell hybridoma cells coexpressing mouse CD3 with human Jurkat TcR α and β chains. The shortage of monoclonal antibodies (mAbs) specific for the human TcR-Vα and Vβ families reflects the difficulties in their production by conventional methods using whole human T cells or purified soluble receptors as immunogens. As an alternative strategy to circumvent these difficulties, we have generated a transfected mouse T-cell line expressing a human (Jurkat) TcR αβ dimer in a complex with mouse CD3. The parental mouse T-cell line, TG40, is a cell surface TcR-negative, cytoplasmic CD3-positive variant of the mouse T-cell hybridoma 2B4. The human-TcR αβ expressing mouse transfectant was used to immunize mice with the same genetic background as the parent mouse T-cell line, and a human TcR-specific response was successfully achieved. MAb-producing hybridomas were generated by fusing spleen cells from the immunized mice with the mouse myeloma cell line NSO. Of 124 hybridoma supernatants screens, 72 showed reactivity to the human T-cell line Jurkat. Twenty-four of the hybridomas producing human (Jurkat) TcR-specific antibodies were cloned and screened for reactivity to Jurkat TcR. Several IgG2b and IgM mAbs specific for the Jurkat T cell line were selected on the basis of their ability to modulate surface CD3 expression on Jurkat cells. Most of the antibodies do not stain other TcR-expressing human T cell leukemia cell lines, implying specificity for the variable domains of the Jurkat TcR. Each of the mAbs reacted with a minor population of peropheral blood lymphocytes (2%–6%), further indicating specificity for the variable domains of the TcR. Of the eight mAbs that were studied in detail, two appeared to have specificity for the Vβ8 TcR family, as judged by their reactivity with T-cell lines, and the increase in Vβ8 + T cells induced by culturing human peripheral blood mono-nuclear cells in the presence of the antibody. By using the system developed here, full-length human TcRα and TcRβ cDNA clones made from polyclonally activated human peripheral blood T-lymphocytes will be transfected into the TcR-negative mouse T-cell line for the production of a panel of human TcR-specific monoclonal antibodies. These would be invaluable reagents for the study of the human TcR repertoire in health and disease.

Cocaine augments proliferation of human peripheral blood T-lymphocytes activated with anti-CD3 antibody

Previously we observed that cocaine can suppress the phytohemagglutinin-induced proliferation of cultured, purified human T-lymphocytes. However, because the receptor activation pathways stimulated by this mitogen are not fully understood, we decided in the present study to examine the effects of cocaine on T-lymphocyte cultures stimulated with the anti-CD3 antibody which is known to stimulate these cells through the T-cell receptor complex. The results show that cocaine augments T-lymphocyte proliferation to anti-CD3 stimulation at drug concentrations observed in the blood of cocaine abusers. This augmentation was dose dependent reaching a plateau at a concentration above 0.75 μM. The amount of interleukin-2 (IL-2), as measured by ELISA, in the supernatants of T-cell cultures and the level of cytosolic free-calcium (Ca 2+) mobilization in the T-cells were also increased at approximately the same concentrations that increased proliferation. Cocaine treatment alone had no effects on proliferation, IL-2 production, or Ca 2+ mobilization. These results suggest that cocaine augments proliferation of human T-lymphocytes when the cells are activated thorugh the T-cell receptor complex by increasing cytosolic Ca 2+ mobilization and subsequent IL-2 production.

A quantitative assay for measuring the induction of mutations in human peripheral blood T-lymphocytes

We optimized conditions for propagating freshly-isolated human peripheral blood T-lymphocytes and cells that had been stored in liquid nitrogen on Day 5 post-isolation, exposing them to mutagens in exponential growth, and measuring the cytotoxicity of the agent from the loss of colony-forming ability, and its mutagenecity from the increase in frequency of 6-thioguanine-resistant cells. Supernatant containing T-cell growth factor, from 60Co-irradiated peripheral mononuclear cells cultured in the presence of 60Co-irradiated B-lymphoblastoid human cells as allogeneic stimulators, supplied at a concentration of 10% along with 10% serum and 10 5 allogeneic stimulator cells/ml, supported exponential growth (population doubling times of 22 h) for extended periods (> 30 d). It gave cloning efficiencies of ≥ 40%. T-lymphocytes stored in liquid nitrogen and returned to culture shortly before mutagen exposure exhibited the same sensitivity as freshly-isolated T-cells to killing by the agents tested, i.e., UV radiation, ethylnitrosourea, and (±)-7 β,8 θ-dihydroxy-9 α,19 α,epoxy-7,8,9,10-tetrahydrozobenzo[ a]pyrene. We showed that if mutagenized polupations frozen during the expression period thawed and assayed, they exhibited the same cloning efficiencies and frequencies of 6-thioguanine-resistant cells as did the corresponding populations that had been assayed directly without freezing. Use of these procedures should facilitate investigation of the frequency and kinds of mutations induced in the HPRT gene of peripheral blood T-lymphocytes in vivo and in vitro.

Southern-blot analyses of human T-lymphocyte mutants induced in vitro by γ-irradiation

G 0 phase cultures of human peripheral blood T-lymphocytes from a single individual were exposed to 300 rad of γ-irradiation from a 137Cs source and cultured in vitro for 8 days to allow phenotypic expression. Thioguanine-resistant (TG r) mutants were isolated by a cell cloning assay in microtiter plates. These mutants were studied by Southern blot analysis to define the gross structural alterations in the hypoxanthine-guanine phosphoribosyl transferase ( hprt) gene by use of an hprt cDNA probe. A similar analysis of the T-cell receptor (TCR) gene rearrangement patterns was employed to define the independent nature of each mutant colony by use of TCR β and γ cDNA probes. 74 mutants were isolated in 5 separate experiments. TCR gene rearrangement analysis showed these to represent 24 independent mutations, of which 18 contained hprt structural alterations. These alterations included simple deletions ( 10 18 ) as well as more complex rearrangements resulting in molecular weight changes of restriction fragments representing both the 5′ and 3′ regions of the hprt gene ( 4 18 and 4 18 , respectively). These results demonstrate that γ-irradiation primarily induces TG r mutations through gross structural alterations in the hprt gene and that these alterations are randomly distributed across the gene. This approach to mutation analysis will provide information on the types of alterations induced by this irradiation, especially the extent of deletions involving the hprt gene. These results also demonstrate the feasibility of employing in vitro exposure of human T-lymphocytes to a single mutagenic agent as an aid to understanding the mechanisms of mutations occurring in vivo in humans.

The metabolism of platelet-activating factor in human T-lymphocytes

The metabolism of 1-[ 3H]alkyl-2-acetyl- sn-glycero-3-phosphocholine (1-[ 3H]alky1-2-acetyl-GPC; platelet-activating factor; PAF) was investigated in purified human peripheral blood T-lymphocytes and a human leukemia cell line of T-cell origin (MOLT-4). The major metabolic products of T-lymphocyte PAF metabolism are 1-alky1-2-acyl-GPC, 1-alkyl-2-lyso-GPC and neutral lipid. The pattern of PAF metabolism in peripheral blood T-lymphocytes and MOLT-4 lymphoblasts was similar, although MOLT-4 lymphoblasts transformed PAF to 1-alky1-2-acyl-GPC faster than peripheral blood T-lymphocytes (67% vs. 21% of added label after 64 min at 37°C, respectively). Pre-exposure of MOLT-4 lymphoblasts to 1 mM of the serine hydrolase inhibitor phenylmethylsulfonyl fluoride resulted in an inhibition of PAF metabolism. Our results indicate that intact T-lymphocytes actively metabolize this biologically active phospholipid by the deacetylation-transacylation pathway.

High pressure liquid chromatography of cyclic nucleotide phosphodiesterase from purified human T-lymphocytes

The cyclic nucleotide phosphodiesterase (EC 3.1.4.17) in extracts of purified human peripheral blood T-lymphocytes was examined by ion exchange high pressure liquid chromatography. Four peaks of activity were isolated. The first peak of activity selectively hydrolyzed cyclic GMP. The following 3 peaks of activity (Ia, IIa and IIIa) were selective for cyclic AMP. The selective low K m cyclic AMP-phosphodiesterase inhibitor, Ro 20-1724 (d,l-1,4-[3-butoxy-4-methoxybenzyl]-2-imidazolidinone), did not inhibit the activity in Ia whereas it did inhibit the activity in IIa and IIIa (IC 50 = 17 μM). The authors conclude that ion exchange high pressure liquid chromatography described in this communication is a useful method for the isolation of different forms of cyclic nucleotide phosphodiesterase activity from human T-lymphocytes.

Enhancement of the Expression of Activation Markers on Human Peripheral Blood Mononuclear Cells by In Vitro Culture With Retinoids and Carotenoids

Retinoids (retinol, retinal, retinoic acid, retinyl beta-glucuronide, and 13-cis retinoic acid) and carotenoids (beta-carotene and canthaxanthin) were evaluated for their immunomodulatory effects on human peripheral blood T-lymphocyte subpopulations and natural killer (NK) cells. Peripheral blood mononuclear cells (PBMC) from healthy young volunteers were isolated and incubated for 72 hours at various levels of retinoids and carotenoids including a physiological concentration (10(-8) M). Expression of surface antigens for total T cells, T-helper and T-suppressor cells, and activation markers (transferrin receptor, HLA-Dr antigen, and interleukin 2 receptor) were analyzed with an EPICS V flow cytometer. Retinoic acid and 13-cis retinoic acid (13-cRA) produced significant increases in the percentage of cells with markers for total T-helper cells, with a minimal effect on percentage of lymphocytes with markers for NK cells. However, beta-carotene (BC), canthaxanthin (CTX), and retinyl beta-glucuronide (RBG) dramatically increased the percentage of PBMC with markers for NK cells and produced a smaller increase in lymphocytes with surface antigens identifying them as T-helper cells. Furthermore, retinol and retinal did not show significant change either in the percentage of lymphocytes with markers for T-helper cells or in the helper/suppressor ratio. An increase in the expression of HLA-Dr antigen and transferrin receptors was greater when cells were incubated with 13-cRA than with either BC, CTX, or RBG, while carotenoids produced a greater increase in the expression of IL-2 receptors than 13-cRA. Our study indicates that both retinoids and carotenoids might be activating different subpopulations of immune cells.

Phosphatidyl inositol hydrolysis after CD3 binding in human peripheral blood T cells: Inhibition by prostaglandin E 2

Hydrolysis of phosphatidyl inositol-4,5-biphosphate, leading to generation of inositol phosphates (IPs), occurs after crosslinking CD3 antigens on the surface of murine and human T-cell lines and clones, and is thought to represent a basic mechanism of signal transduction after antigen receptor binding. Previous investigators have had difficulty demonstrating this phenomenon using human peripheral blood T-cells. In this paper we demonstrate significant IP generation after anti-CD3 stimulation of human peripheral blood lymphocytes and T-cells. The amount of IP generation is less and considerably more variable than what is obtained using a T-cell line. Also, exposure to ammonium chloride in the E-rosetting procedure totally inhibits IP generation, perhaps explaining previous unsuccessful attempts. Finally, prostaglandin E2 and other agents that raise cyclic AMP inhibit IP generation by anti-CD3 antibodies in human T-cells and a T-cell line.

Quantitation and Cloning of Human Urushiol Specific Peripheral Blood T-Cells: Isolation of Urushiol Triggered Suppressor T-Cells

A limiting dilution assay was developed to quantitate urushiol (the antigen of poison ivy; Toxicodendron radicans) specific T cells from peripheral blood of a patient with a history of rhus (poison ivy) dermatitis. It was found that maximal sensitivity with minimal nonspecific proliferation could be produced with the use of 5 U/ml of recombinant IL2 added to the assay on day 6. This donor was found to have a frequency of urushiol specific peripheral blood T cells of (1/2935). Five interleukin 2 (IL2) dependent urushiol specific T-cell clones were generated from the peripheral blood of this patient. These T-cell clones had a CD8+ (T8+) phenotype and proliferated specifically to both extracts of Toxicodendron radicans (poison ivy) leaves and pure urushiol. Pentadecylcatechol was an inferior antigen, only stimulating proliferation of one clone. The ability of all clones to proliferate to pure urushiol, despite their having been induced with leaf extract, suggests that urushiol, or closely related catechols, represent the only allergenic constituents of Toxicodendron radicans. Lymphokine production in response to antigen varied between (0.6-5.0) units/ml of interleukin 2 (IL2) and (1.0-120) units/ml of gamma interferon. Although none of the clones showed significant cytotoxicity against NK targets, three of five lines showed considerable cytotoxicity against concanavalin A treated (lectin approximated) targets. However, cytotoxicity for rhus conjugated autologous targets was not detected. It was found that several of these CD8+ clones could suppress IgG production in the presence of rhus antigen. The isolation of these T-cells from peripheral blood several months after rhus dermatitis suggests that these clones may have a role in down regulating delayed hypersensitivity to urushiol.