Regulation of interleukin-2 and interleukin-6 production from T-cells: involvement of interleukin-1 beta and transforming growth factor-beta

The effect of recombinant (r) interleukin-1 beta (rIL-1 beta) and transforming growth factor-beta (TGF-beta) on the production of interleukin-2 (IL-2) and interleukin-6 (IL-6) from an antigen-specific (LBRM-33-1A5) and an antigen-nonspecific (EL-4-NOB-1) T-cell line was investigated. rIL-1 beta induced the production of IL-2 and IL-6 from EL-4-NOB-1 cells in a dose-related manner. The LBRM-33-1A5 cells required phytohemagglutinin (PHA) in addition to rIL-1 beta in order to produce IL-2 and IL-6. IL-2 production was found to precede IL-6 production in both cell lines. No IL-2 or IL-6 production was observed by adding r murine tumor necrosis factor-alpha or r murine interferon gamma to the cells. The presence of 1 ng/ml TGF-beta reduced IL-2 and IL-6 production from both T-cell lines by more than 80%. The inhibition of IL-2 and IL-6 production was still evident by a concentration as low as 10 pg/ml of TGF-beta. rIL-1 beta and PHA also stimulated murine thymocytes to produce IL-6 which was inhibited up to 85% in the presence of 1 ng/ml TGF-beta. Taken together these results suggest that TGF-beta may suppress immune responses by inhibiting the endogenous production of IL-2 and IL-6.     

Endogenous production of prothymocyte differentiating activity by phytohemagglutinin-stimulated T-cell-depleted human marrow

The PHA responsiveness of marrow T-cell precursors remains a matter of controversy. We have investigated the capacity of human marrow to proliferate under phytohemagglutinin (PHA) stimulation following extensive removal of mature T cells by complement-dependent cytotoxicity with MBG6 and RFT8 monoclonal antibodies. PHA-induced thymidine uptake by marrow cells occurred with a peak on Days 6-8 of incubation instead of Day 3 for PBL. This peak was observed 48 hr earlier in the presence of PHA-stimulated T-depleted marrow cell supernatants. These supernatants can also promote the growth of mature T-cell colonies from MBG6-, RFT8-, T11-, T3- marrow. However, full colony development requires exogenous interleukin 2 (IL-2). IL-2 could be detected in marrow supernatants but only at very low levels and beyond Days 3 and 4. In contrast Days 1-6 marrow supernatants were equally effective in promoting MBG6-RFT8- marrow cell responsiveness to PHA. We conclude that marrow T-cell precursors are not PHA responsive and that PHA induces the production by marrow non-T cells of a prothymocyte-differentiating activity (PTDA); PTDA can differentiate marrow T-cell progenitors into PHA-responsive T cells; following activation by PHA, these cells undergo limited proliferation induced by IL-2 endogenously released from de novo differentiated T cells. It is suggested that this mechanism may account for extrathymic differentiation of the T-cell lineage in heavily irradiated marrow transplantation recipients.     

Signal requirements for lymphocyte activation: Role of a T-cell growth factor produced by guinea pig peritoneal exudate lymphocytes

Peritoneal exudate lymphocytes (PEL) from immunized guinea pigs, when pulsed with antigen, rapidly release a T-cell stimulatory factor (TSF). TSF nonspecifically enhances the proliferation of purified guinea pig T cells in the presence of another signal such as PMA, PHA, or Con A. On a per cell basis, antigen-pulsed PEL produce about 14 times more activity than similarly stimulated lymph node lymphocytes.Several lines of evidence support the view that TSF is the guinea pig equivalent of TCGF (IL-2). TSF containing supernatants have IL-2 activity when assayed on the IL-2-dependent CT6 cell line. When TSF containing supernatants were absorbed with CT6 cells, there was a significant decrease of both TSF activity as assessed on guinea pig T cells as well as IL-2 activity as assessed by the CT6 assay. Additionally, partially purified human and mouse IL-2 have TSF activity, while the macrophage product, IL-1, has no TSF activity. After chromatography on a S-200 column, TSF activity and IL-2 activity coelute at an apparent molecular weight of 19,000.     

Cyclosporin A blocks calcium-dependent pathways of gene activation.

We have used an interleukin-2 (IL-2) promoter-CAT fusion gene to study activation of IL-2 gene expression by IL-1, phytohemagglutinin (PHA), phorbol myristate acetate (PMA), and calcium ionophore in the murine thymoma line EL4 and the human lymphoma line Jurkat. The two cell lines respond differently to combinations of these stimuli. IL-1 in combination with suboptimal concentration of PMA induced chloramphenicol acetyltransferase (CAT) activity in EL4. In Jurkat cells, IL-1 failed to synergize with PMA or PHA. Cotransfection with the IL-2/CAT gene and a construct capable of expressing murine T-cell type IL-1 receptors converted Jurkat cells to IL-1 responsiveness. IL-1 in combination with PHA but not with PMA resulted in induction of CAT activity in these cells. Induction of IL-2/CAT activity by all stimuli in both cell lines was blocked by the presence of EGTA in the culture medium. EGTA did not inhibit IL-1/PMA activation of an SV40 early promoter-CAT fusion gene in either EL4 or Jurkat cells; therefore, calcium was not required for IL-1 or PMA signal transduction. Jurkat cells were shown to differ from EL4 in their requirement for calcium mobilization. Two different calcium-dependent pathways of gene activation were distinguished, both of which were blocked by the immunosuppressive drug cyclosporin A.     

Aberrant activation of the interleukin-2 autocrine loop through the nuclear factor of activated T cells by nonleukemogenic human T-cell leukemia virus type 2 but not by leukemogenic type 1 virus

Human T-cell leukemia virus type 1 (HTLV-1) but not HTLV-2 is associated with adult T-cell leukemia. We found that HTLV-2 Tax2 protein stimulated reporter gene expression regulated by the interleukin (IL)-2 promoter through the nuclear factor of activated T cells (NFAT) in a human T-cell line (Jurkat). However, the activity of HTLV-1 Tax1 was minimal in this system. T-cell lines immortalized by HTLV-2 but not HTLV-1 constitutively exhibited activated NFAT in the nucleus and constitutively expressed IL-2 mRNA. Cyclosporine A, an inhibitor of NFAT activation, abrogated the induction of IL-2 mRNA in HTLV-2-immortalized T-cell lines and concomitantly inhibited cell growth. This growth inhibition was rescued by the addition of IL-2 to the culture. Furthermore, anti-IL-2 receptor antibodies significantly reduced the proliferation of HTLV-2-infected T-cell lines but not that of HTLV-1-infected cells. Our results suggest that Tax2 activates an IL-2 autocrine loop mediated through NFAT that supports the growth of HTLV-2-infected cells under low-IL-2 conditions. This mechanism would be especially important in vivo, where this autocrine mechanism establishes a nonleukemogenic life-long HTLV-2 infection. The results also suggest that differences in long-term cytokine production between HTLV-1 and HTLV-2 infection are another factor for the differences in pathogenesis.     

Accessory cell-derived helper signals in human T-cell activation with phytohemagglutinin: induction of interleukin 2-responsiveness by interleukin 6, and production of interleukin 2 by interleukin 1 [corrected

Interleukins (IL-) 1 and 6 have been shown to represent accessory signals for T-cell activation. In the present study, we further examined the effects of both cytokines on accessory cell-depleted human T cells stimulated with phytohemagglutinin (PHA). The addition of IL-6 to the cultures resulted in T-cell proliferation; however, IL-1 was unable to support PHA-induced T-cell growth. The addition of IL-1 consistently induced a low level of IL-2 production and strongly enhanced T-cell proliferation in the presence of IL-6. Thus, the effect of IL-1 on T-cell growth becomes apparent only in the presence of IL-6. Blocking the IL-2-receptor (IL-2R) with the monoclonal antibodies anti-Tac and MikBêta 1 (directed to the alpha and bêta chains of the IL-2R, respectively) had no effect on PHA/IL-6-supported proliferation, but completely eliminated the growth-enhancing effect of IL-1. On the other hand, a neutralizing anti-IL-4-antiserum did not affect PHA/IL-6- or PHA/IL-6/IL-1-induced proliferation. Further experiments showed that IL-6 enhances T-cell responsiveness to IL-2, as evidenced by enhanced IL-2-induced proliferation. However, we could not find an effect of IL-6 on the expression of IL-2R as measured by staining with anti-Tac and with MikBêta 1 or by binding of (125I)-IL-2 to T cells. It can be concluded from these studies that IL-1 and IL-6 have different helper effects on PHA-induced T-cell activation. In the presence of PHA, IL-6 induces limited IL-2/IL-4-independent growth, and more importantly it renders T cells responsive to IL-2. IL-1 provides a signal leading to IL-2 production. The combination of IL-1 and IL-6 represents a synergistic helper signal, leading to an IL-2-dependent pathway of proliferation.     

IL-2 modulation of murine T-cell oncogene expression

C-myb, a cellular oncogene associated with normal thymic development, was found to be highly expressed in four interleukin 2 (IL-2)-independent T-cell lines, but not in two of three IL-2-dependent T-cell lines. The IL-2-dependent lines, HT2 and CTLL-2, were found to have low levels of c-myb mRNA in the presence of IL-2. However, short-term IL-2 depletion resulted in at least fivefold increases in c-myb message. Add-back of IL-2 after 30 hr IL-2 depletion of CTLL-2 cells resulted in return to baseline low-level c-myb mRNA. Expression of the oncogenes myc, bas, raf, and abl as well as the T-cell genes Thy-1 and CT beta did not parallel that of c-myb. These studies indicate that removal of a growth factor can result in increased levels of a specific cellular oncogene and that two nuclear protooncogenes (c-myb and c-myc) are expressed differentially during cell growth. These results may help to explain aspects of intrathymic T-cell differentiation where there is very high c-myb expression in the face of limiting amounts of growth factors such as IL-2.     

Proliferative response of normal and activated human lymphocytes to tetradecanoyl phorbol acetate

The mitogenicity of 12-O-tetradecanoyl phorbol-13-acetate (TPA) for normal human peripheral blood mononuclear cells was investigated. TPA was a weak mitogen giving simulation indices in the range 2.5 to 10.5 at the optimum concentration (10 ng/ml) compared with 39 to 95 for phytohemagglutinin (PHA) at its optimum concentration (1 μg/ml). No absolute requirement for a comitogen could be demonstrated, however TPA and PHA were synergistic in their action at low concentrations, and additive at optimum concentrations. Cell fractionation by rosetting with sheep erythrocytes showed that most of the proliferative response to TPA occurred in the T-cell fraction, however some proliferation of non-T cells was also observed. Surface marker studies showed that this could not have been due to residual T cells in the non-T fraction. A small number of monocytes was required for optimal proliferation of T cells in response to TPA. After a 3-day incubation with mitogen, the responding cell populations were tested for binding of a range of antibodies specific for T-cell (OKT3, OKT4, OKT8, and OKT11), “natural killer” (NK) cell (anti-Leu-7), monocyte (FMC17), and B-cell (anti-human immunoglobulin) surface markers. These experiments indicated that the responding cell types were T cells and B cells, but not NK cells or monocytes. Marked modulation of the antigen detected by OKT4, and to a lesser extent that detected by OKT3, in the presence of TPA precluded determination of which subpopulations of T cells proliferated in response to TPA. TPA was also tested for its ability to “maintain” activated T-cell blasts in a standard assay for interleukin 2 (IL-2). Mitogen-activated T cells were strongly responsive to TPA in this assay, but progressively lost responsiveness when maintained in crude IL-2 for about 2 weeks. Thus TPA does not have “maintenance” (i.e., IL-2-like) activity. However, small amounts of TPA acted synergistically with PHA in maintaining blast populations which were not responsive to TPA alone. This illustrates the importance of using long term IL-2-dependent cell lines for quantitation of IL-2 in supernatants prepared by stimulating T cells with these agents.     

Activated lck tyrosine protein kinase stimulates antigen-independent interleukin-2 production in T cells.

p56lck, a member of the src family of cytoplasmic tyrosine kinases, is expressed predominantly in T cells where it associates with the T-cell surface molecules CD4 and CD8. Mutants of CD4 and CD8 that have lost the ability to associate with p56lck no longer enhance antigen-induced T-cell activation. This suggests that p56lck plays an important role during T-cell activation. In an effort to understand the function of p56lck in T cells, a constitutively activated lck gene (F505lck) was introduced into T-helper hybridoma cell lines by retroviral infection. In four T-cell lines we examined, the activated lck protein stimulated interleukin-2 (IL-2) production, a hallmark of T-cell activation, in the absence of antigenic stimulation. In addition, a marked increase in antigen-independent IL-2 production was apparent when T cells infected with a temperature-sensitive F505lck were shifted to the permissive temperature. Only one cell line expressing F505lck exhibited increased sensitivity to antigenic stimulation. The SH3 domain of p56lck was dispensable for the induction of antigen-independent IL-2 production. In contrast, deletion of the majority of the SH2 domain of p56F505lck reduced its ability to induce spontaneous IL-2 production markedly. Activated p60c-src also induced antigen-independent IL-2 production, whereas two other tyrosine kinases, v-abl and the platelet-derived growth factor receptor, did not. Tyrosine phosphorylation of a 70-kDa cellular protein was observed after cross-linking of CD4 in T cells expressing F505lck but not in cells expressing F527src.     

Differential signal requirements in T-cell activation by mitogen and superantigen

The requirement for co-stimulatory molecules in T-cell stimulation by mitogens and superantigens in the absence of antigen-presenting cells (APC) was investigated. Phytohemagglutinin (PHA) induced interleukin (IL)-2 receptor (IL-2R) expression on purified T-cells, but proliferation occurred only when exogenous IL-2 was added. In contrast, the proliferative response to a pepsin-extracted type 5 M-protein from Streptococcus pyogenes (pep M5), a recently identified superantigen, required signals provided by phorbol 12-myristate 13-acetate (PMA), IL-1 and IL-6. pep M5 alone did not induce IL-2R expression; however, when combined with PMA, IL-1 and IL-6, IL-2R was expressed. Differences were also observed in the response of the leukemic T-cell line, Jurkat, to PHA and pep M5. Soluble PHA, but not pep M5, induced IL-2 production by these cells in the presence of PMA. Cross-linking by its specific antibody or adsorption of pep M5 to microtiter plates was required to activate Jurkat cells. Both PHA and pep M5 induced Ca²⁺ mobilization in Jurkat cells; however, only PHA induced a rise in intracellular Ca²⁺ in purified T-cells, whereas pep M5 was unable to induce this activity unless IL-1, IL-6 and PMA were added. Our data provide biochemical evidence that mitogenic and superantigenic stimulation of T-cells is different.     

T-cell activation is required for efficient replication of human herpesvirus 6.

We have investigated whether T-cell activation is required for the replication of the T-lymphotropic human herpesvirus 6. The virus did not replicate in quiescent peripheral blood lymphocytes but replicated efficiently following exposure of the cells to the polyclonal mitogen phytohemagglutinin (PHA). When purified T cells were treated with PHA in the absence of accessory cells, no virus replication was observed unless exogenous interleukin-2 (IL-2) was added to the medium, promoting cell division. Incubation of peripheral blood lymphocytes in the absence of PHA but in the presence of IL-2 resulted in delayed cell blastogenesis and virus replication. Cell blastogenesis and virus replication did not occur in the purified T-cell cultures incubated with IL-2 alone. Taken together, the results show that human herpesvirus 6 replication requires full progression of the cell cycle. This finding might have implications for the pathogenicity of the virus in the human host.     

Development of a serum-free medium which supports the long-term growth of human and nonhuman primate lymphoid cells

The ability to grow lymphoid cells in serum-free media affords the advantage of separately analyzing those components found to be involved in proliferation and differentiation. Iscove's medium (IMDM) supplemented with bovine serum albumin or casein, cholesterol, ferrous chloride, insulin, β-mercaptoethanol, L-α-phosphatidylcholine, and transferrin supported the long-term proliferation of a gibbon ape lymphoma T-cell line, MLA144. These cells continue to produce Interleukin 2 (IL-2, T-cell growth factor) constitutively in the serum-free medium. IL-2-dependent human T cells initiated and maintained in culture in serum-free medium containing IL-2 have continued to replicate for over 3 months with two population doublings every 3 to 4 days. A normal, IL-2-dependent marmoset T-cell line, OH-1, also proliferated on the serum-free medium when supplemented with IL-2. Several established primate B-cell lines which do not require IL-2 for growth were able to proliferate in the serum-free medium. These B-cell lines included B95-8, an Epstein-Barr virus (EBV)-transformed marmoset cell line, HuCo/R-H, a human cord B-lymphocyte line transformed with EBV, and Namalwa, an EBV-positive B-cell line established from a Burkitt's lymphoma. B95-8 cells grown on serum-free medium showed high levels of EBV antigen-positive cells after induction with 12-O-tetradecanoyl-phorbol-13-acetate (TPA).     

Characterization of interleukin 2-dependent cytotoxic T-cell clones. IV. Production of alpha, beta and gamma interferons and interleukin 2 by Lyt-2+ T cells

The production of alpha, beta and gamma interferons (IFN) and interleukin 2 (IL-2) by Lyt-2+-dependent cytotoxic T-cell lines/clones was investigated. Cloned and uncloned T-cell lines specific for H-2Dd or the unique RL male 1 leukemia antigen were studied. After infection with Sendai virus (SV) or Newcastle disease virus (NDV) all cell lines produced IFN-alpha and -beta. Induction of IFN-gamma was attempted with the mitogens Con A, PHA, PWM, SEA, and SEB, with poly(I:C), with antibodies Lyt-1.2, -2.2, and Thy-1.2, or with the target cells Meth A (H-2Dd+) and RL male 1. All mitogens were effective inducers. However, the antibodies and poly(I:C) were not. One uncloned RL male 1-specific cell line CTLL-RP, produced IFN-gamma after induction with RL male 1. Production of IFN-alpha, beta depended on IL-2, whereas production of IFN-gamma did not, although addition of highly purified IL-2 increased IFN-gamma production even in the absence of other inducers. Crude IL-2 inhibited the production of IFN-gamma but not IFN-alpha, beta. In response to mitogens, some T-cell clones also produced IL-2. The results demonstrate that Lyt-2+ cells can produce a broad spectrum of lymphokine activities after appropriate stimulation. Their availability now affords us the opportunity to study the regulation of lymphokine production at the clonal level.     

Immunomodulating activity in supernatants from EBV immortalized lymphocytes

Summary Our earlier work has demonstrated that EBV immortalized B lymphocytes are involved in a factor dependent autostimulatory cycle. Soluble growth stimulating activity was released into culture supernatants by these growing B cells. Growth enhancing (GE) media from B lymphocyte lines, immortalized by EBV infection, contained soluble factor(s) which modulated the Con A response of normal human mononuclear cells. Conditioned media from these lines affected the Con A response in a biphasic manner, stimulating the blastogenic response at lower concentrations, while inhibiting at higher concentrations. At stimulatory concentrations, the blastogenic response to Con A began earlier than in controls and was markedly enhanced by day 2. GE media reduced the initial response of purified B cells to pokeweed mitogen. GE media did not support growth of IL-2 dependent cells. GE media from some EBV-carrying B cell lines had measurable IL-1 activity in the mouse thymocyte PHA response. GE media from LPS stimulated B lymphocyte lines produced significant IL-1-like effects on stimulated mouse thymocytes. These results suggested that these B cell lines may produce IL-1-like factors that cooperate in T cell responses. The possibility that such factors may play a role in B lymphocyte transformation by EBV is discussed.Supported by grants from the Concern Foundation and the Brigham Surgical Foundation     

Differential requirement for accessory cells in polyclonal T-cell activation

Highly purified rat Ia-negative (OX-6-) and Ia-positive (OX-6+) T cells were employed to examine the requirement for accessory cells (AC) and/or soluble factors in the activation of resting T cells with Con A, PHA, sodium periodate, or antigen. A variety of cells were employed as AC, including Ia-positive and Ia-negative macrophages (M phi), gamma-irradiated (2000 rad) or non-irradiated OX-6+ T cells, and several Ia-negative adenovirus-transformed rat embryo fibroblast cell lines. Our results suggested that for the expression of IL-2 receptors (IL-2R) and proliferation of OX-6- T cells in response to Con A, PHA, or antigen, there was an obligatory requirement for the presence of AC which could not be overcome by the addition of IL-1 and/or IL-2. Activation of OX-6- T cells with antigen required the presence of Ia+ AC, while activation with mitogens could be initiated with Ia- AC. M phi were efficient in AC function in all responses tested, while the AC function of OX-6+ T cells (TAPC) proved discriminatory under different conditions. The optimal response to PHA required much higher concentrations of TAPC as AC than for the Con A response. TAPC failed to stimulate sodium periodate-treated T cells under any conditions tested. Furthermore, when TAPC were employed as AC, their antigen-presenting ability was radiosensitive, while their AC function for Con A and PHA was radioresistant. These results suggest that molecules involved in T cell-AC interactions may differ, depending on the source of AC and/or type of the proliferative stimulus provided to T cells. This data has been discussed in the context of T-cell activation.     

Human T lymphocyte activation in the presence of acute myelogenous leukaemia blasts; studies of normal polyclonal T cells and T lymphocyte clones derived early after allogeneic bone marrow transplantation

 T cell clones (CD4⁺CD8^–TCRαβ⁺γδ^–) derived from bone marrow transplant recipients were stimulated with phytohaemagglutinin (PHA) +interleukin-2 (IL-2) in the presence of irradiated (50 Gy) peripheral blood mononuclear cells (PBMC) derived from acute leukaemia patients(leukaemic PBMC containing more than 95% blast cells). Leukaemic PBMC could function as accessory cells during mitogenic T cell activation resulting in both T cell proliferation and a broad T cell cytokine response [IL-3, IL-4, IL-10, granulocyte/macrophage-colony-stimulating factor (GM-CSF) tumour necrosis factor α (TNFα) and interferon γ (IFNγ) secretion]. Blockade of IL-1 effects by adding IL-1 receptor antagonist together with PHA+IL-2+leukaemia blasts increased T cell proliferation, whereas IL-6-neutralizing antibodies did not alter T cell proliferation. A qualitatively similar T cell cytokine response and a similar cytokine profile (highest levels detected for GM-CSF and IFNγ) were detected when normal polyclonal T cell lines were stimulated with PHA in the presence of non-irradiated leukaemic PBMC. When leukaemic PBMC derived from 18 acute myelogenous leukaemia patients were cultured with PHA and cells from a polyclonal T cell line, increased concentrations of the T cell cytokines IFNγ and IL-4 were detected for all patients. We conclude that T cell activation resulting in proliferation and a broad cytokine response can take place in the presence of excess acute myelogenous leukaemia blasts. Received: 30 November 1995 / Accepted: 9 January 1996     

Inhibition of lymphocyte proliferation by monoclonal antibody directed against the T3 antigen on human T cells

Peripheral blood mononuclear cells from 40% of normal donors are mitogenically unresponsive to UCHT1, a monoclonal antibody reactive to the T3 surface molecule on human T lymphocytes. Cell preparations from non-UCHT1 responders were used to examine whether and how interaction of UCHT1 with the T3 molecule affects T-cell functionality. It was found that UCHT1 profoundly (greater than 85%) suppressed lymphocyte proliferation induced by plant mitogens (phytohemagglutinin (PHA) and concanavalin A (Con A], recall antigen (candidin), and allogeneic non-T cells. The antibody abrogated both the production of interleukin 2 (IL-2) by and the expression of IL-2-specific receptors on T lymphocytes stimulated by PHA or allogeneic non-T cells. UCHT1 was maximally suppressive when added to cells within 2 hr (PHA stimulation) or 1 day (allogeneic non-T cell activation) after the initiation of the culture period. The inhibiting activity of UCHT1 could be related to its ability to modulate T3 molecules from the T-cell surface: both actions displayed the same antibody concentration dependence and had a comparable time dependence. Moreover, after modulation, unresponsive lymphocytes regained responsiveness to PHA in parallel with reexpression of surface T3 molecules. These findings are consistent with the idea that the human T3 molecule functions as an essential signal transducer during the early phases of T-cell activation.     

Interleukin-18 enhances the production of interferon-gamma (IFN-gamma) by allergen-specific and unspecific stimulated cord blood mononuclear cells

BACKGROUND: IL-18 is a pleiotropic cytokine involved in the polarisation of T-cell response. This study was performed to determine whether or not IL-18 is detectable in phytohemagglutinin (PHA) or betalactoglobulin (BLG) stimulated supernatants of cord blood mononuclear cells (CBMC) and to study the in vitro effect of IL-18 on the interferon (IFN)-gaamma and IL-13 release of CBMC of healthy neonates. METHODS: CBMC of neonates were isolated by Ficoll density centrifugation. The cytokines IFN-gamma, IL-13 and IL-18 in the cell culture supernatants were measured using the ELISA technique following stimulation with a unspecific (PHA 20 microg/ml) and an allergen-specific stimulus (BLG 25 microg/ml). In order to study the in vitro effect of IL-18, CBMC were stimulated either with medium alone or with IL-18, IL-18 + PHA and IL-18 + BLG. RESULTS: IL-18 levels in supernatants of CBMC were low and did not vary significantly between unstimulated and PHA or BLG stimulated cell cultures (median 21.4; 23.5 and 15.5 pg/ml, respectively). IFN-gamma and IL-13 levels were significantly higher in response to PHA and BLG (PHA: IFN-gamma, 6154; IL-13, 4357; BLG: IFN-gamma, 801; IL-13, 249 pg/ml) compared to unstimulated cell cultures. The addition of IL-18 to PHA or BLG stimulated CBMC significantly enhanced the IFN-gamma release (PHA: 6154; PHA + IL-18: 13474, p = 0.0001; BLG: 801; BLG + IL-18: 1077, p = 0.008). In comparison to incubation without IL-18, the release of IL-13 was invariable or even reduced, when CBMC were stimulated with PHA + IL-18 (4026, p = 0.16) or BLG + IL-18 (124, p = 0.0001) compared to stimulation of CBMC with PHA (4357 pg/ml) or BLG (249 pg/ml) alone. CONCLUSIONS: IL-18 is detectable in supernatants of CBMC. We observed a significant effect of IL-18 + PHA as well as IL-18 + BLG on IFN-gamma release in vitro. Based on our findings we conclude that IL-18 could act as a strong TH1-inducing factor on stimulated CBMC also in vivo.     

Suppression of interleukin-2 and interleukin-2 receptor expression in Jurkat cells stably expressing the human immunodeficiency virus Tat protein.

The Jurkat T cell line was stably transfected with an Epstein-Barr virus-based episomal replicon designed to express high levels of the HIV-1 Tat protein. After selection in hygromycin B, high-level Tat activity was detected in 3 of 18 transfected cell lines. After stimulation with phytohemagglutinin (PHA) and phorbol myristate acetate (PMA), Tat transfectants with high Tat expression showed diminished expression of interleukin-2 (IL-2) and the interleukin-2 receptor alpha chain (IL-2R) when compared to untransfected Jurkat cells or Jurkat cell lines transfected with the parent control plasmid. Sublines derived from the high-level Tat transfectants with reduced Tat activity showed normalization of PHA/PMA-induced IL-2 expression. Northern analysis showed diminished expression of IL-2 and IL-2R mRNA in the stimulated Tat transfectants. Inhibition of IL-2 and IL-2R expression by the HIV-1 Tat protein may contribute to the immune suppression that characterizes HIV-1 infection.     

Human acute lymphoblastic leukemia (ALL) blasts as accessory cells during T-cell activation: differences between patients in costimulatory capacity affect proliferative responsiveness and cytokine release by activated T cells

The ability of acute lymphoblastic leukemia (ALL) blasts to mediate costimulatory signals during T-lymphocyte activation was investigated in an experimental model in which monoclonal T-cell populations were stimulated with standardized activation signals (anti-CD3 and anti-CD28 monoclonal antibodies; phytohemagglutinin, PHA). Leukemia cells from 12 consecutive ALL patients with high peripheral blood blast counts were studied. Proliferative T-cell responses were detected for a majority of these patients when irradiated leukemia blasts were used as accessory cells during activation. T-cell cytokine release was also observed for most patients when using nonirradiated ALL accessory cells. Low or undetectable cytokine levels were usually observed for CD8+ clones, whereas the CD4+ clones often showed a broad cytokine response with release of interleukin-2 (IL-2), IL-4, IL-10, IL-13 and interferon gamma(IFN-gamma) in the presence of the ALL accessory cells. ALL blasts were also able to function as allostimulatory cells for normal peripheral blood mononuclear responder cells. However, both T-cell proliferation and cytokine release showed a wide variation between ALL patients. The accessory cell function of ALL blasts showed no correlation with the release of immunomodulatory mediators (IL-2, IL-10, IL-15) or the expression of any single adhesion/costimulatory membrane molecule (CD54, CD58, CD80, CD86) by the blasts. We conclude that for a majority of patients, native ALL blasts can mediate costimulatory signals needed for accessory cell-dependent T-cell activation, but differences in costimulatory capacity between ALL patients affects both the proliferative responsiveness and cytokine release by activated T cells.