Growth of an interleukin 2/interleukin 4-dependent T cell line induced by granulocyte-macrophage colony-stimulating factor (GM-CSF)

Lymphokine activities in conditioned medium from activated helper T cell lines are most commonly defined by the proliferation of "specific" lymphokine-dependent cell lines. Various sublines of IL 2-dependent (and ostensibly specific) HT-2 and CTLL cells have now been shown to proliferate in response to BSF-1/IL 4 as well. After activation with antigen or mitogen, D10.G4.1, an antigen-specific cloned T helper cell that has recently been shown to produce IL 4 but not IL 2, secretes two distinct cytokines that induce the growth of HT-2 cells. These "T cell growth factors" (TCGF) can be separated by reversed phase high-performance liquid chromatography (RP-HPLC). The TCGF activity of one of these factors can be blocked by 11B11, an antibody specific for IL 4. The second TCGF activity is not affected by 11B11 or by antibodies specific for IL 2. This TCGF activity can be neutralized by a goat polyclonal antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF), and has a RP-HPLC elution profile identical to that of recombinant GM-CSF. Recombinant GM-CSF induces both proliferation and long-term growth of HT-2 but not CTLL cells, and this activity can be neutralized by the same antibody to GM-CSF. GM-CSF is best known as a factor that induces the maturation and growth of granulocytes and macrophages from bone marrow-derived hematopoietic precursor cells. The ability of GM-CSF to induce the growth of certain T cell lines indicates that this molecule may play a role in T cell-mediated immune responses, either as an autocrine growth factor or a paracrine stimulus from both lymphoid and nonlymphoid tissues that produce this cytokine.     

Cloned, Ia-restricted T cells that do not produce interleukin 4(IL 4)/B cell stimulatory factor 1(BSF-1) fail to help antigen-specific B cells

T cells can be subdivided based on cell surface markers, MHC restriction, function, and production of soluble factors. Analysis of the ability of cloned, Ia-restricted, L3T4+ T cells to induce an in vitro anti-hapten antibody response to hapten-carrier conjugates allowed the definition of three functional subtypes. To examine whether these functional subtypes also differed in the production of soluble mediators, supernatants of the cloned lines were examined for the production of T cell growth factors and factors inducing increased expression of Ia glycoproteins on small resting B cells. All of the cloned lines produced T cell growth factors that could be further differentiated by inhibition with monoclonal antibodies. None of the Ia-restricted, L3T4+ cloned T cell lines that failed to produce IL 4/BSF-1 could provide helper function. Thus, the activation of antigen-specific B cells by helper T cells appears to require IL 4/BSF-1 as a necessary but not sufficient signal for differentiation into antibody-forming cells.     

Characterization of a keratinocyte-derived T cell growth factor distinct from interleukin 2 and B cell stimulatory factor 1

Epidermal epithelial cells (keratinocytes) produce and secrete a variety of immunologically active cytokines. We have previously reported that both transformed (PAM 212) and normal murine keratinocytes produce a soluble factor which induces proliferation of the T cell line, HT-2. In the present study we sought to compare keratinocyte-derived T cell growth factor (KTGF) with other T cell growth factors, characterize its physicochemical properties, and substantially purify KTGF from PAM 212 conditioned medium. KTGF from PAM 212 conditioned medium was not inhibited by antibodies which block the effect of interleukin 2 (IL 2) (S4B6) or B cell stimulatory factor 1 (BSF 1) (11B11). KTGF is heat-stable, has an isoelectric point of 4.8, and a relative molecular mass of 16 to 23 kilodaltons under nonreducing conditions. KTGF activity was enhanced at least 41,413-fold by sequential hydroxylapatite bulk preparation, desalting by reversed-phase chromatography, gel filtration high pressure liquid chromatography (HPLC), and reversed-phase HPLC. Keratinocytes produce a T cell growth factor with physicochemical properties distinct from IL 2 and BSF 1. KTGF may play a role in regulating the growth and differentiation of T cells in the epidermis.     

Characterization of a T4+/Leu-8+ T cell clone that directly helps B cell Ig production by secreting B cell differentiation factor

A human T4+/Leu-8+ T cell clone (YA2) was established by phytohemagglutinin activation and interleukin 2 (IL 2) propagation. Functional characterization of this clone demonstrated that it provided potent help towards Ig production by pokeweed mitogen-stimulated B cells in the presence of small numbers of autologous T cells or by Staphylococcus aureus Cowan I (SAC)-activated B cells in the presence of B cell growth factor (BCGF). YA2 provided no help to resting B cells and minimal help to either unactivated B cells cultured with BCGF or SAC-activated B cells. Supernatant generated from clone YA2 by IL 2 stimulation had significant B cell differentiation activity but no BCGF or IL 2 activity. Thus, YA2 is a T4+/Leu-8+ potent direct helper only to B cells that are activated and proliferating due to its selective secretion of a differentiation factor, and not an activation and growth factor. The availability of phenotypically defined cloned populations of T cells with restricted functional helper activity related to the secretion of selected B cell tropic factors should prove useful in the dissection of the role of individual T cell subsets in the regulation of the human B cell cycle.     

Species-specificity of T cell stimulating activities of IL 2 and BSF-1 (IL 4): comparison of normal and recombinant, mouse and human IL 2 and BSF-1 (IL 4)

Mouse and human interleukin 2 (IL 2) both cause proliferation of T cells of the homologous species at high efficiency. Human IL 2 also stimulates proliferation of mouse T cells at similar concentrations, whereas mouse IL 2 stimulates human T cells at a lower (sixfold to 170-fold) efficiency. In contrast, the T cell stimulating activities of mouse and human B cell stimulatory factor 1 (interleukin 4; IL 4) appear to be species specific over the range of concentrations tested; we detected no activity of mouse IL 4 on human T cells, or human IL 4 on mouse T cells.     

Synergistic and antagonistic effects of recombinant human interleukin (IL) 3, IL-1 alpha, granulocyte and macrophage colony-stimulating factors (G-CSF and M-CSF) on the growth of GM-CSF-dependent leukemic cell lines

Three human leukemia cell lines (TALL-101, AML-193, and MV4-11) that require granulocyte/macrophage-colony stimulating factor (GM-CSF) for growth in a chemically defined medium were examined for their response to recombinant human (rh) cytokines. Either rh interleukin (IL)-3 or rhGM-CSF alone supported the long term growth of all three cell lines, and the two growth factors acted synergistically to stimulate the proliferation of the early T lymphoblastic leukemia (TALL-101) and of the monocytic leukemia (AML-193) cells. However, IL-3 antagonized the proliferation of the biphenotypic B-myelomonocytic leukemia (MV4-11) cells in the presence of GM-CSF when both factors were used at very low concentrations. The rh granulocyte (G)-CSF independently supported the long and short term growth of AML-193 and MV4-11, respectively, and synergized with GM-CSF in inducing proliferation of these cells. By contrast, G-CSF did not stimulate TALL-101 cell growth and antagonized the effect of GM-CSF such that proliferation was arrested. Although neither rh macrophage (M)-CSF nor rhIL-1 alpha independently promoted proliferation of the three leukemia cell lines, these cytokines were able to either up- or down-regulate the GM-CSF-dependent growth of these cells. Taken together, these data demonstrate that leukemic cells often require the synergistic action of several cytokines for optimal growth, whereas other combinations of factors may be growth-inhibitory. This raises the possibility that multiple hemopoietic growth factors sustain or control leukemic cell proliferation also in vivo. In addition, the observation the G-CSF, M-CSF, and IL-1 alpha can, in some cases, arrest cell proliferation without inducing differentiation suggests that the programs of proliferative arrest and differentiation in leukemic cells can be dissociated.     

Interleukin 2-mediated immune interferon (IFN-gamma) production by human T cells and T cell subsets

Human interleukin 2 (IL 2, or T cell growth factor), which was free of lectin and interferon activity (IFN), induced human peripheral T lymphocytes to produce immune IFN (IFN-gamma). In contrast, non-T cells and macrophages did not produce IFN-gamma in response to IL 2. IL 2 acted directly on unstimulated T cells to induce IFN-gamma production, and also acted in synergy with a suboptimal dose (2 micrograms/ml) of concanavalin A (Con A) to enhance IFN-gamma production. The IFN-gamma-inducing activity of partially purified IL 2 was absorbed along with the IL 2 activity by murine IL 2-dependent CT-6 cell line cells. This further supports the view that IFN-gamma-inducing activity is identical to IL 2. When T cells were separated further into helper/inducer T4+ and suppressor/cytotoxic T8+ subsets by negative selection with monoclonal antibody and complement, both T4+ and T8+-enriched cells produced significant levels of IFN-gamma in response to IL 2. Complete removal of macrophages from purified T lymphocyte populations by treatment of OKM1 plus complement consistently reduced IFN-gamma production in response to IL 2 to a limited degree; readdition of macrophages restored IFN-gamma production by both T cell subsets. This observation that IL 2 contributes to the production of IFN-gamma by human lymphocytes suggests that a cascade of lymphocyte-cell interactions participates in human immune responses.     

Effect of infection with murine recombinant retroviruses containing the v-src oncogene on interleukin 2- and interleukin 3-dependent growth states

The cloned murine interleukin 3 (IL 3)-dependent cell lines FD.C/1, 32Dc1-23, and KP3 can each be switched to interleukin 2 (IL 2)-dependent growth states. Replication-defective retroviral vectors have been used to introduce the v-src oncogene into each of these cell lines maintained in either an IL 3- or an IL 2-dependent growth state. These cell lines maintained in an IL 3-dependent growth state were converted to lymphokine-independent growth after infection with v-src. These same cells maintained in an IL 2-dependent growth state and infected with v-src maintained strict lymphokine dependence for growth. Another cloned murine IL 3-dependent cell line, GM, can be switched to a granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent growth state. GM cells maintained as IL 3- or GM-CSF-dependent cells readily converted to a lymphokine-independent growth state when infected with v-src. These experiments indicate that either there exist differences in the biochemical mechanisms of signal transduction through the IL 3- and IL 2-specific receptors, or developmental processes associated with the switching of cells to an IL 2-dependent growth state influence expression of the v-src gene product. These cell lines offer new ways not only for analyzing biochemical pathways that regulate cell growth, but also for analyzing the control of oncogene expression.     

A helper factor needed for the generation of mouse cytolytic T lymphocytes is made by tumor cell lines, cloned T cells, and spleen cells exposed to a variety of stimuli

A helper factor termed cytolytic T lymphocyte helper factor (CHF) that is needed for the generation of allospecific mouse cytolytic T lymphocytes (CTL) in vitro was produced by mouse spleen cells 3 to 4 days after the time when interleukin 2 (IL 2) had reached its maximal production. These kinetics were observed by stimulation of immune spleen cells with allogeneic tumor or spleen cells, with Sendai or influenza viral peptides, with virus infected cells, or with concanavalin A (Con A). CHF produced by rat spleen cells was able to help in the generation of mouse CTL, indicating that this cytokine was not restricted genetically. CHF could also be made by WEHI-3 and EL4 cell lines, as well as cloned cytolytic and helper T cells. The production of CHF by WEHI-3 cells argues that CHF is not IL 2. In addition, if CHF was not present early in the in vitro stimulation no CTL were generated, suggesting that CHF participated in the activation of CTL precursors. The addition of IL 2-containing conditioned medium to the CHF assay resulted in no substantial CTL generation, although significant cellular proliferation was observed. In contrast, CHF-containing conditioned medium allowed the generation of CTL in the absence of the same level of proliferation.     

Differential effects of interleukin-2 and interleukin-4 on protein tyrosine phosphorylation in factor-dependent murine T cells

Interleukin-2 (IL-2) is a requisite factor for growth and proliferation of IL-2-dependent T cells. At present, the mechanism by which the high-affinity IL-2-IL-2 receptor interaction transmits a mitogenic signal to the cellular interior remains unclear. In this report we have used three murine T cell clones to demonstrate that IL-2 stimulates rapid tyrosine phosphorylation of several proteins. Two of these clones, CTLL-2 and CT6, exhibit a cytotoxic T cell phenotype, while the third, HT-2, was derived from a helper T cell line. All three T cell clones proliferated in response to IL-2 stimulation, but HT-2 cells also proliferated in response to interleukin-4 (IL-4). We comparatively examined the effects of IL-2 and IL-4 on protein tyrosine phosphorylation in these cells by immunoaffinity purification of phosphotyrosyl substrates with an anti-phosphotyrosine monoclonal antibody. Stimulation with concentrations of IL-2 resulting in maximal (10-30 U/ml) or sub-maximal (1-5 U/ml) proliferation caused the rapid tyrosine phosphorylation of 97 and 57 kDa proteins in all three cell lines. The 97 kDa protein was localized in the cytosol, while the 57 kDa protein was detected in both cytosolic and crude membrane fractions. IL-2-dependent tyrosine phosphorylation of an 86 kDa cytosolic protein was observed only in CT6 cells. Tyrosine phosphorylation of 22, 23 and 200 kDa proteins was also observed, but only in the cytotoxic T cell clones. Phosphoamino acid analyses revealed that the 97, 86 and 57 kDa proteins contained phosphotyrosine and phosphoserine residues. Concentrations of IL-2 below the threshold concentration for induction of a proliferative response correspondingly failed to stimulate protein tyrosine phosphorylation. In contrast, growth stimulation of HT-2 cells by IL-4 was not preceded by early changes in protein tyrosine phosphorylation, suggesting that protein tyrosine phosphorylation may not be essential for the induction of IL-4-dependent cell-cycle progression. These results demonstrate that high-affinity IL-2 receptors are coupled to tyrosine kinase activity(s) in T cells. However, the failure of IL-4 to stimulate protein tyrosine phosphorylation in the same cells indicates that enhanced protein tyrosine phosphorylation may not be requisite for growth factor-dependent T cell proliferation.     

T cell growth factors from adult T cell leukemia virus-transformed cell lines

Some characteristics of T cell growth factors derived from adult T cell leukemia virus (ATLV)-transformed cell lines, MT 1 and MT 2 were analyzed. MT 1 cells release significant interleukin 2 (IL 2) activity into the culture medium, which showed the same elution pattern of gel filtration and isoelectric focusing of IL 2 from lectin-stimulated normal human lymphocytes. This activity was also detected in the cell extract of MT 1. In contrast, MT 2 cell line did not produce IL 2 activity, but non-IL 2 type growth factor was observed. The significance of these factors from MT cell lines is discussed from the viewpoint of 'autokine' in ATLV-transformed cells.     

Characterization of a novel murine T cell-activating factor

Purified resting peripheral lymph node T cells can be activated to produce interleukin 2 (IL 2) and to proliferate in the presence of Concanavalin A (Con A) and an apparently novel lymphokine that we call T cell activating factor (TAF). TAF is biochemically distinct from IL 1, IL 2, IL 3, and other colony stimulating factors, IL 4 (BSF-1) and interferons. Furthermore, of the recombinant and natural cytokines tested, only IL 2 and TAF are active in the TAF assay. In the presence of Con A, TAF stimulates an increase in the steady-state level of IL 2 mRNA in T cells, the secretion of active IL 2 into the culture medium, and the proliferation of the T cells. We propose that TAF is a previously undescribed molecule the function of which is to stimulate IL 2 production by T cells that have encountered antigen, and we propose that TAF has an important role in primary T cell immune responses.     

Biological and biochemical characterization of bovine interleukin 2. Studies with cloned bovine T cells

Bovine peripheral blood lymphocytes stimulated with the T cell mitogen concanavalin A (Con A) secrete a lymphokine with biological properties similar to T cell growth factor (TCGF) or interleukin 2 (IL 2) from other species. The material supports proliferation of Con A-derived T cell blasts, limiting dilution cloning of T cell blasts, and continuous growth of T cell clones for over 6 mo in vitro. A quantitative microassay with the use of TCGF-dependent, Con A-unresponsive cloned T cells was used to determine the biological activity during purification of IL 2. A single peak of activity with an apparent m.w. of 25,000 to 28,000 was recovered after gel filtration. This material eluted from DEAE-Sephacryl between 135 and 165 mM NaCl. After isoelectric focusing, high pressure liquid chromatography, and gel electrophoresis under reducing conditions, peak IL 2 activity was associated with proteins having m.w. of 20,000 and 23,000.     

The role of interleukin 2 and T11 E rosette antigen in activation and proliferation of human NK clones

Although considerable data have recently been accumulated regarding the functional role of natural killer (NK) cells, relatively little is known about the factors that regulate NK cell activity. In these studies, we evaluated the role of interleukin 2 (IL 2) and the expression of the IL 2 receptor in the activation and proliferation of human NK cloned cell lines. By using a series of cloned cell lines, we were able to analyze homogeneous populations of NK cells that ordinarily comprise only a small fraction of peripheral blood lymphocytes and are extremely heterogeneous with respect to phenotypes and cytotoxic specificities. In comparison with several T cell clones, we found a much lower density of IL 2 receptors on NK clones, regardless of whether or not these cloned cells had a mature T cell phenotype. Correspondingly, NK clones needed a 10-fold higher concentration of recombinant IL 2 for maximal proliferation. Moreover, blocking studies with specific monoclonal IL 2 receptor antibodies indicated that IL 2 is both necessary and sufficient to induce the proliferation of NK clones. Because the majority of peripheral blood NK cells and NK clones express the T11 E rosette receptor antigen, which has been shown to be an antigen-independent activation pathway for T cells, we were able to study the role of monoclonal anti-T11 antibodies in the activation of various NK clones for which a specific target antigen is not known. In contrast to T cell clones, the induction of IL 2 receptor expression after T11 activation was possible only for some NK clones such as JT10 and JT3, but not for CNK5. Before activation, the IL 2 receptor expression of NK clones was confined to cells in the G2 - M phase, but after T11 activation the more pronounced IL 2 receptor expression became independent of the cell cycle. With respect to the direct proliferative effect of anti-T11 activation that has been noted with T cell clones, only the T3+ (JT10) and not the T3- NK clones could be directly stimulated. Nevertheless, IL 2 receptor expression could be triggered on some T3- clones such as JT3. Because T11-induced proliferation of T cells has been shown to be dependent on both the expression of the IL 2 receptor and on the interaction of this receptor with IL 2, it is proposed that the different responses of NK cells to T11 activation may reflect the ability of the individual clone to produce endogenous IL 2, as well as its ability to express the IL 2 receptor.     

Antibodies to the L3T4 and Lyt-2 molecules interfere with antigen receptor-driven activation of cloned murine T cells

When L3T4+ cloned murine helper T lymphocytes (HTL) are stimulated with antigen or immobilized anti-T cell receptor (TCR) monoclonal antibodies (mAb) at concentrations which are optimal for proliferation, anti-L3T4 mAb inhibits activation as measured by proliferation and lymphokine production. Under similar conditions, IL 2-independent proliferation of Lyt-2+ cloned murine cytolytic T lymphocytes (CTL) stimulated by anti-TCR mAb is inhibited by anti-Lyt-2 antibodies. Proliferation of cloned HTL and CTL cells stimulated by IL 2 is not affected by the anti-L3T4 and anti-Lyt-2 mAb. The inhibition of TCR-induced activation of the T cell clones is not due to interference with the binding of the anti-TCR mAb. Stimulation of the TCR has been proposed to induce lymphokine secretion and proliferation by T cells through a pathway involving the activation of protein kinase C and the stimulation of an increase in the concentration of intracellular free calcium. However, proliferation of T cells stimulated by PMA (which activates protein kinase C) plus the calcium ionophore A23187 (which increases the concentration of intracellular free calcium) is not affected by mAb reactive with the Lyt-2 or L3T4 structures. If TCR stimulation does indeed activate T cells by activating protein kinase and increasing intracellular free calcium, then our data suggest that anti-L3T4 and anti-Lyt-2 mAb inhibit TCR-driven proliferation at some step before the activation of protein kinase C and the stimulation of a rise in intracellular free calcium concentration. Our results suggest that anti-L3T4 and anti-Lyt-2 mAb interfere with early biochemical processes induced by stimulation of the TCR. In HTL, which proliferate via an autocrine pathway, anti-L3T4 mAb appears to inhibit proliferation by interfering with signaling events involved in lymphokine production. Inhibition of IL 2-independent proliferation of Lyt-2+ cells by anti-Lyt-2 mAb appears to occur by a different mechanism. The precise molecular basis for the interference of each cell type has not yet been characterized.     

Production of a fibroblast-stimulating factor by Schistosoma mansoni antigen-reactive T cell clones

Fibrosis in schistosomiasis is the terminal event of a complex pathophysiologic cascade involving interactions between fibroblasts and both host and parasite products. In the present study, the effect of lymphokines produced by cloned Schistosoma mansoni antigen-reactive T cells on the proliferation of murine fibroblasts was investigated. These T cells previously have been shown to proliferate, produce lymphokines, mediate delayed-type hypersensitivity responses, and generate in vitro granulomas in response to soluble egg antigen (SEA). T cells, co-cultured with irradiated antigen-presenting cells and pulsed with SEA, produced levels of fibroblast-stimulating factor (FSF) comparable to equivalent numbers of dispersed hepatic granuloma cells isolated from infected mice. Supernatants of cloned T cells pulsed with Con A (in the absence of macrophages) contained no detectable interleukin 1 activity, but did stimulate fibroblast activation and growth. T cell FSF activity was trypsin-sensitive, was stable at 56 degrees C but not to boiling, and was retained by Con A Sepharose. Activity was associated with HPLC fractions corresponding to an m.w. of 10,000 to 40,000. Neither recombinant interferon-gamma nor affinity-purified interleukin 2 was capable of stimulating fibroblast proliferation. In functional studies, the degree of fibroblast proliferation was related to the length of exposure to the factor. In addition, quiescent fibroblasts were maximally stimulated by T cell FSF only if a second co-factor such as insulin or epidermal growth factor was present. The synergism between T cell FSF and known progression factors suggests that FSF-T may provide a competence signal to fibroblasts. The present results suggest that a direct molecular link may exist between T cells and fibroblasts in schistosomiasis.     

PMA alone induces proliferation of some murine T cell clones but not others

The responses of cloned murine T cell lines to the phorbol ester, phorbol myristate acetate (PMA), were investigated. PMA alone was able to stimulate proliferation of some clones but not others. Two Lyt-2+, cloned cytolytic T lymphocyte (CTL) lines proliferated in response to stimulation by PMA alone, but several L3T4+, cloned helper T lymphocyte (HTL) lines did not. In contrast, all clones tested released lymphokines in response to stimulation by the combination of PMA and the calcium ionophore A23187. Moreover, all clones proliferated in response to stimulation by the combination of PMA and A23187. The proliferation of HTL in response to PMA + A23187 could be completely inhibited either by cyclosporine A (CsA) or by PC61.5, a monoclonal antibody directed against the murine IL 2 receptor; however, the proliferation of CTL in response to PMA alone was not affected either by CsA or by PC61.5. These results suggest that of the murine T cell clones tested, HTL proliferate in response to stimulation via an IL 2-dependent, autocrine pathway; in contrast, CTL, in addition to an IL 2-dependent pathway, may possess an additional IL 2-independent pathway of proliferation. CTL that proliferate in response to stimulation by PMA alone may be useful models in the study of T cell proliferation.     

Retrovirus infection alters growth factor responses of T lymphocytes

A murine helper/inducer T cell clone, D10.G4, has been infected with Kirsten-murine sarcoma virus (KiSV) pseudotyped with an amphotropic murine leukemia virus. The resultant Ki-ras-expressing lines (KiSV-D10) remain dependent on exogenous factors for continued growth but display distinctly different mitotic responses to certain cytokines as compared to the uninfected parent clone. Unlike the parent D10.G4 cells, these KiSV-D10 cells can be maintained in vitro indefinitely in the presence of recombinant interleukin 2 (IL 2), and they all display a maximal proliferative response to purified or recombinant interleukin 1 (IL 1). The IL 1-induced proliferation is shown not to be dependent or secretion of the T cell autocrine growth factors IL 2 or B cell stimulatory factor-1 (BSF-1). The KiSV-D10 lines show certain differences from one another and parent D10.G4 cells in their secretory and proliferative responses to T cell receptor- and BSF-1 mediated signals. These viral oncogene-expressing T cell lines, which remain responsive to and dependent on physiologic growth factors, should prove valuable for analyzing the mechanisms of action of single oncogenes and the intracellular events in T lymphocyte activation.     

Prostaglandin E2-dependent induction of granulocyte-macrophage colony-stimulating factor secretion by cloned murine helper T cells

PG are known to inhibit T cell proliferation, at least in part by suppressing IL-2 production, but effects of PG on the production of other lymphokines have not been well studied. We have found that PGE2 and PGE1, but not PGF2 alpha, inhibit both proliferation and production of granulocyte-macrophage (GM)-CSF by murine TH clones stimulated with Ag or anti-CD3 antibody. Thus, signals generated via the Ag receptor:CD3 complex were inhibited by PGE. Most interesting, however, was the finding that PGE2 and PGE1 could act synergistically with IL-2 for the induction of GM-CSF in some TH1 clones. Dependence on PGE2 for this response was not found in all clones, as some TH1 cells could produce GM-CSF after IL-2 alone, and some cells did not produce GM-CSF even in the presence of PGE2 and IL-2. These observations indicate that there is a subset of TH1 cells receptive to a stimulating activity of PGE2 in the presence of IL-2. PGE2 is known to elevate cAMP levels in T cells. Therefore, we tested whether other agents known to increase cAMP, such as forskolin and cholera toxin, could act in conjunction with IL-2 to induce GM-CSF secretion. As was found with PGE2, these compounds also induced GM-CSF activity in the presence of IL-2, suggesting a critical role for cAMP in this process. Overall these data indicate that the requirements for activation of GM-CSF secretion vary among individual T cells. Most importantly they provide the first evidence that E-series PG are positive signals for lymphokine induction in certain T cells, whereas simultaneously acting as negative signals limiting proliferation. This result also suggests that treatment with anti-inflammatory drugs that decrease PGE2 concentrations may inhibit lymphokine secretion normally stimulated by this pathway.     

Generation and characterization of cloned T helper cell lines for anti-DNA responses in NZB.H-2bm12 mice.

We have previously demonstrated that the introduction of the bm12 mutation into NZB mice results in animals that spontaneously produce high titer IgG autoantibodies to dsDNA. The observation that NZB.H-2bm12 develop lupus although NZB.H-2b control mice do not, provides a unique system to study the role of Th cells in the production of antibodies to dsDNA. We have isolated, in the absence of a known stimulating autoantigen, a series of seven autoreactive T cell clones that provide help in vitro for the production of IgG anti-dsDNA antibodies by syngeneic B cells. The data on these seven cloned T cell lines was compared to two cloned T cell lines specific for keyhole limpet hemocyanin. The seven cloned T cell lines, coined clones 19D, 23G, 410F, 410H, C1, C15, and C52 all show significant help in vitro for production of IgM and IgG antibodies to ssDNA and dsDNA; antibody levels increased 7- to 30-fold compared to cultures without T cells. Clones C1, C15, and C52 were furthered studied and were shown to provide help for IgM antihistone and anti-OVA responses but provided significantly less help for IgG antibodies. In contrast, keyhole limpet hemocyanin-specific cloned T cell lines TK2 and TK5 provided help for IgM antibodies to ssDNA, dsDNA, and histone, but failed to significantly increase IgG antibodies to ssDNA, dsDNA, or histone. The cloned T cell lines were restricted to H-2bm12 and proliferated only in response to APC from NZB.H-2bm12 and B6.C-H-2bm12 but not NZB.H-2b or NZB.H-2d mice; their in vitro helper activity was inhibited by antibodies to class II. All cloned T cell lines expressed Thy-1, CD5, and TCR-alpha/beta. Three of the seven clones used TCR-V beta 4. However, the V beta expression of the four remaining autoreactive T cell clones could not be determined. All of the autoreactive cloned T cell lines produce significant IL-4 but no detectable IL-2 or IFN-gamma. We believe that HPLC-purified peptides eluted from I-Abm12 molecules from APC can potentially provide insight on the putative autoantigen.