Biosynthesis and posttranslational regulation of human IL-12.

IL-12 is a heterodimeric proinflammatory cytokine consisting of a light alpha-chain, formerly defined as p35, disulfide-linked to a heavier beta-chain, formerly defined as p40. The beta-chain is also produced in large excess in a free form, and disulfide-linked beta-chain homodimers with anti-inflammatory effects are produced in the mouse. We analyzed the biosynthesis and glycosylation of IL-12 in human monocytes, and in a cell line stably transfected with IL-12 alpha and beta genes (P5-0.1). The IL-12 heterodimer and free beta-chain were immunoprecipitated from supernatants and cell lysates of metabolically labeled cells and resolved in SDS-PAGE. Whereas the beta-chain showed similar pI pattern whether in the free form or associated in the heterodimer, either in the secreted or intracellular form, the alpha-chain in the secreted heterodimer was much more acidic than that present in the intracellular heterodimer. Deglycosylation experiments with neuraminidase and Endo-F combined with two-dimensional PAGE of single bands of the intracellular vs extracellular IL-12 heterodimer revealed that the alpha-chain was extensively modified with sialic acid adducts to N-linked oligosaccharides before secretion. N-glycosylation inhibition by tunicamycin (TM) did not alter free beta-chain secretion, while preventing the IL-12 heterodimer assembling and secretion. Pulse-chase experiments indicated that IL-12 persists intracellularly for a long period as an immature heterodimer, and that glycosylation is the regulatory step that determines its secretion. beta-chain disulfide-linked homodimers were observed in TM-treated P5-0.1 cells, but in neither TM-treated nor untreated monocytes.

CD4(+) T cell clones producing both interferon-gamma and interleukin-10 predominate in bronchoalveolar lavages of active pulmonary tuberculosis patients.

The pattern of cytokine production in T cell clones derived from bronchoalveolar lavages (BAL) of active pulmonary tuberculosis (TB) patients was analyzed in clones obtained by limiting dilution procedures which expand with high efficiency either total T lymphocytes, independently of their antigen-recognition specificity, or Mycobacterium tuberculosis-specific T cells. BAL-derived clones, representative of CD4(+) cells from five patients with active TB, produced significantly higher amounts of IFN-gamma than BAL-derived CD4(+) clones from three inactive TB donors or four controls (with unrelated, noninfectious pathology). Average IL-4 and IL-10 production did not differ significantly in the three groups. Although these data suggest a predominant Th1 response to M. tuberculosis infection in the lungs, the majority of BAL-derived CD4(+) clones produced both IFN-gamma and IL-10 and the percentage of clones with this pattern of cytokine production was significantly higher in clones derived from BAL of active TB patients than from controls. Only rare clones derived from peripheral blood (PB)-derived CD45RO(+) CD4(+) T cells of both patients (nine cases) and controls (four cases) produced both IFN-gamma and IL-10; instead, the IL-10-producing clones derived from PB T cells most often also produced IL-4, displaying a typical Th2 phenotype. Higher average amounts of IFN-gamma and IL-10 were produced by BAL-derived CD8(+) clones of four active TB patients than of four controls, although the frequency of CD8(+) clones producing both IFN-gamma and IL-10 was lower than that of CD4(+) clones. The M. tuberculosis-specific BAL-derived T cell clones from three active TB patients were almost exclusively CD4(+) and produced consistently high levels of IFN-gamma often in association with IL-10, but very rarely with IL-4. Unlike the BAL-derived clones, the M. tuberculosis-specific clones derived from PB CD45RO(+) CD4(+) T cells of three different active TB patients and two healthy donors showed large individual variability in cytokine production as well as in the proportion of CD4(+), CD8(+), or TCR gamma/delta(+) clones. These results indicate the predominance of CD4(+) T cells producing both the proinflammatory cytokine IFN-gamma and the anti-inflammatory cytokine IL-10 in BAL of patients with active TB.

Differential susceptibility to HIV-GP120-sensitized apoptosis in CD4+ T-cell clones with different T-helper phenotypes: role of CD95/CD95L interactions.

The susceptibility of Th1 and Th2 cell clones to apoptosis following HIV-gp120/CD4 cross-linking and TCR activation was investigated. We show that only Th1 clones are susceptible to HIV-gp120-sensitized apoptosis, although both types of clones express similar levels of CD4 and bind similar amounts of recombinant gp120. Both types of clones, however, undergo apoptosis induced by CD95 cross-linking with agonistic monoclonal antibody (MoAb). Apoptosis induced by gp120 in the Th1 clones is inhibited by either an anti-CD95 neutralizing MoAb or an anti-CD95L neutralizing MoAb as well as by a specific interleukin-1 beta converting enzyme (ICE) inhibitor. When triggered to apoptosis by gp120, Th1 but not Th2 clones express both cell-associated and soluble CD95L. The CD95L produced by Th1 clones induces cell death, inhibitable by anti-CD95 neutralizing MoAb, of CD95 positive Jurkat cells. These data suggest that, like activation-induced apoptosis, HIV-gp120 sensitized apoptosis in Th1 clones occurs via CD95/CD95L interaction and that lack or insufficient production of CD95L is responsible, at least in part, for the resistance of Th2 clones to such apoptosis.

Acute induction and priming for cytokine production in lymphocytes.

When T-lymphocytes (CD4+, CD8+, or TCR gamma delta +) and NK cells proliferate in vivo or in vitro in response to exposure to antigen or other stimuli, they often segregate into subsets with the ability to produce either type-1 [interferon-gamma (IFN-gamma) and interleukin-2 (IL-2)] or type-2 cytokines (IL-4, IL-5, IL-6 and IL-10). IL-12 induces the differentiation of type-1 cytokine-producing T-cells primarily through its ability to prime them for high IFN-gamma production; however, paradoxically IL-12 also primes T-cells for high production of the type-2 cytokine IL-10. Priming of T-cells for IL-4 production requires the presence of IL-4, but it is maximally observed in cultures containing both IL-4 and IL-12. IL-12, in addition to priming T-cells for high IFN-gamma and IL-10 production, is also a potent acute inducer of expression of the IFN-gamma gene in T- and NK-cells, and, to a much lower extent, of the IL-10 gene. IL-4, which has a very powerful effect in priming T-cells for IL-4 production, does not appear to have a significant ability to directly activate the expression of the IL-4 gene. Thus, IL-12 and IL-4 affect the expression of type-1 and type-2 cytokine genes by two different mechanisms: an acute induction of gene expression which is rapid and reversible, and a priming of the genes to a highly responsive state to restimulation, a state that is stable and probably irreversible.

Interleukin-12 primes human CD4 and CD8 T cell clones for high production of both interferon-gamma and interleukin-10.

Interleukin-12 (IL-12) induces differentiation of T helper 1 (Th1) cells, primarily through its ability to prime T cells for high interferon-gamma (IFN-gamma) production. We now report that the presence of IL-12 during the first several days of in vitro clonal expansion in limiting dilution cultures of polyclonally stimulated human peripheral blood CD4+ and CD8+ T cells also induces stable priming for high IL-10 production. This effect was demonstrated with T cells from both healthy donors and HIV+ patients. Priming for IL-4 production, which requires IL-4, was maximum in cultures containing both IL-12 and IL-4. IL-4 modestly inhibited the IL-12-induced priming for IFN-gamma, but almost completely suppressed the priming for IL-10 production. A proportion of the clones generated from memory CD45RO+ cells, but not those generated from naive CD45RO- CD4+ T cells, produced some combinations of IFN-gamma, IL-10, and IL-4 even in the absence of IL-12 and IL-4, suggesting in vivo cytokine priming; virtually all CD4+ clones generated from either CD45RO(-) or (+) cells, however, produced high levels of both IFN-gamma and IL-10 when IL-12 was present during expansion. These results indicate that each Th1-type (IFN-gamma) and Th2-type (IL-4 and IL-10) cytokine gene is independently regulated in human T cells and that the dichotomy between T cells with the cytokine production pattern of Th1 and Th2 cells is not due to a direct differentiation-inducing effect of immunoregulatory cytokines, but rather to secondary selective mechanisms. Particular combinations of cytokines induce a predominant generation of T cell clones with anomalous patterns of cytokine production (e.g., IFN-gamma and IL-4 or IFN-gamma and IL-10) that can also be found in a proportion of fresh peripheral blood T cells with "memory" phenotype or clones generated from them and that may identify novel Th subsets with immunoregulatory functions.

Immunoregulation by interleukin-12.

Interleukin-12 (IL-12) is a heterodimeric cytokine produced primarily by antigen-presenting cells (monocytes, macrophages, dendritic cells, and B cells). Its production is stimulated by bacteria, bacterial products, and intracellular parasites and enhanced by priming with granulocyte-macrophage colony-stimulating factor (CM-CSF) and interferon-gamma (IFN-gamma) or inhibited by IL-10. The major biological activity of IL-12 is on T and natural killer (NK) cells in which it increases cytokine production, proliferation, and cytotoxicity. Its production occurs several hours after exposure to infections agents, which induces a rapid production of IFN-gamma by NK and later by T cells. This IFN-gamma potentiates antigen-presenting cell functions important in clearing infections agents (phagocytosis, oxidative burst, and production of nitrous oxide) and also increases further production of IL-12. IL-12 has been clearly demonstrated to be important in the generation of CD4 and CD8 type 1 T cells both in vivo and in vitro. Our data reveals that IL-12 primes naive T cells for high IFN-gamma and IL-10 production, whereas IL-4 is required for IL-4 priming, thus suggesting that these genes and possibly others are independently regulated. IL-12 is therefore involved in the skewing of cytokine production toward a type 1 and has been implicated in being involved in selective mechanisms of established T cells. It is now becoming clear that the IL-12 acts as both a proinflammatory cytokine and an immunomodulator and therefore bridges the innate and adaptive immune responses.

Selective association of a 22-38 kDa glycoprotein with MHC class II DP antigen on activated human lymphocytes at the plasma membrane.

Two-dimensional electrophoretic analysis (2D-PAGE) of cell surface human DP and DR class II antigens identified a glycoprotein, designated pX, that is associated at the cell surface with DP but not DR class II antigen in activated T, B and NK lymphocytes but not in resting B lymphocytes, Raji B lymphoma cells, activated thymic epithelial cells or activated monocytes. pX is a heavily glycosylated protein with an apparent molecular mass spanning between 38 kDa and 22 kDa, that is reduced, after deglycosylation with Endo-F, to 22 kDa. The pX structure appears nonpolymorphic and independent of DP polymorphism, as suggested by 2D-PAGE migrational pattern of 125I-labelled Endo-F deglycosylated DP immunoprecipitates from T cells blasts derived from four donors with different DP allotypes. The apparent absence of polymorphism of pX is further suggested by two-dimensional peptide mapping of a single spot derived from 2D-PAGE of 125I-labelled DP deglycosylated immunoprecipitates from two donors.

The interleukin 12 p40 gene promoter is primed by interferon gamma in monocytic cells.

Interleukin (IL) 12 is a proinflammatory cytokine produced by phagocytic cells, B cells, and other antigen-presenting cells that modulates adaptive immune responses by favoring the generation of T helper type 1 cells. IL-12 mediates some of its physiological activities by acting as a potent inducer of interferon (IFN) gamma production by T and natural killer cells. IFN-gamma enhances the ability of the phagocytic cells to produce IL-12 and other proinflammatory cytokines. Thus, IL-12-induced IFN-gamma acts in a positive feedback loop that represents an important amplifying mechanism in the inflammatory response to infections. We show here that IFN-gamma enhances IL-12 production mostly by priming phagocytic cells for lipopolysaccharide (LPS)-induced transcription of the IL-12 p40 gene, which encodes the heavy chain of the IL-12 heterodimer; furthermore, IFN-gamma directly induces transcription of the IL-12 p35 gene, which encodes the light chain of IL-12, and has at least an additive effect with LPS stimulation in inducing its transcription. The priming effect of IFN-gamma on the LPS-induced p40 gene transcription requires preincubation of the cells with IFN-gamma for at least 8 h to obtain a maximal effect. The priming effect of IFN-gamma for IL-12 production is predominantly at the transcriptional level for both the p40 and the p35 gene, and no evidence for a major role of posttranscriptional or translational mechanisms was found. A 3.3-kb human IL-12 p40 promoter construct transfected into cell lines recapitulated the tissue specificity of the endogenous gene, being silent in two human T cell lines, constitutively active in two human Epstein-Barr virus-positive B lymphoblastoid cell lines, and LPS inducible in the human THP-1 and mouse RAW264.7 monocytic cell lines. Because the RAW264.7 cell line is easily transfectable and regulates the endogenous IL-12 p40 gene in response to IFN-gamma or LPS similarly to human monocytes, it was used for analysis of the regulation of the cloned human IL-12 p40 promoter. A requirement for the region between -222 and -204 in both LPS responsiveness and IFN-gamma priming was established. This region contains an ets consensus sequence that was shown to mediate activation of the promoter by IFN-gamma and LPS, as well as by a cotransfected ets-2. The -222 construct was also regulated in a tissue-specific manner. Two other elements, IRF-1 located at -730 to -719, and NF-IL6 at -520 to -512, were also studied by deletion analysis, which did not result in decreased response to IFN-gamma and LPS stimulation.

Interleukin 12 induces stable priming for interferon gamma (IFN-gamma) production during differentiation of human T helper (Th) cells and transient IFN-gamma production in established Th2 cell clones.

Interleukin 12 (IL-12) facilitates the generation of a T helper type 1 (Th1) response, with high interferon gamma (IFN-gamma) production, while inhibiting the generation of IL-4-producing Th2 cells in polyclonal cultures of both human and murine T cells and in vivo in the mouse. In this study, we analyzed the effect of IL-12, present during cloning of human T cells, on the cytokine profile of the clones. The culture system used allows growth of clones from virtually every T cell, and thus excludes the possibility that selection of precommitted Th cell precursors plays a role in determining characteristics of the clones. IL-12 present during the cloning procedures endowed both CD4+ and CD8+ clones with the ability to produce IFN-gamma at levels severalfold higher than those observed in clones generated in the absence of IL-12. This priming was stable because the high levels of IFN-gamma production were maintained when the clones were cultured in the absence of IL-12 for 11 d. The CD4+ and some of the CD8+ clones produced variable amounts of IL-4. Unlike IFN-gamma, IL-4 production was not significantly different in clones generated in the presence or absence of IL-12. These data suggest that IL-12 primes the clone progenitors, inducing their differentiation to high IFN-gamma-producing clones. The suppression of IL-4-producing cells observed in polyclonally generated T cells in vivo and in vitro in the presence of IL-12 is not observed in this clonal model, suggesting that the suppression depends more on positive selection of non-IL-4-producing cells than on differentiation of individual clones. However, antigen-specific established Th2 clones that were unable to produce IFN-gamma with any other inducer did produce IFN-gamma at low but significant levels when stimulated with IL-12 in combination with specific antigen or insoluble anti-CD3 antibodies. This induction of IFN-gamma gene expression was transient, because culture of the established clones with IL-12 for up to 1 wk did not convert them into IFN-gamma producers when stimulated in the absence of IL-12. These results suggest that Th clones respond to IL-12 treatment either with a stable priming for IFN-gamma production or with only a transient low level expression of the IFN-gamma gene, depending on their stage of differentiation.

Differential effects of tyrosine kinase inhibition in CD69 antigen expression and lytic activity induced by rIL-2, rIL-12, and rIFN-alpha in human NK cells.

The effect of rIL-12 on induction of CD69 antigen expression and cytolytic activity in purified human NK cells was evaluated in comparison to the effects of rIL-2 and rIFN-alpha. It was found that rIL-12 directly induced CD69 antigen expression in NK cells, although the period of incubation required by rIL-12 was longer than the period required by rIL-2 or by rIFN-alpha. Similarly, the cytolytic activity induced by rIL-12 in NK cells against the NK-resistant target cell line Raji was consistently lower than the cytolytic activity induced by rIL-2 or rIFN-alpha when measured after 6 hr of incubation, and increased during the following 18 hr of incubation. To compare the involvement of tyrosin kinases in activation of NK cells induced by rIL-2, rIL-12, and rIFN-alpha, the effect of the specific inhibitor of tyrosin kinases, genistein, was evaluated on induction of CD69 antigen expression and lytic function mediated by the three cytokines. It was found that genistein inhibited CD69 antigen expression induced by rIL-2 and by rIL-12, but not that induced by rIFN-alpha. Unlike the effect on CD69 antigen expression, the cytolytic activity induced by all three cytokines was inhibited by genistein. These results, together with the finding that CD69 antigen expression induced by rIL-2 but not by rIL-12 or rIFN-alpha was inhibited by addition of rIL-4, strongly suggest that IL-2, IL-12, and IFN-alpha mediate their effects, leading to induction of CD69 antigen expression through different activation pathways.

Different sensitivity to interleukin 4 of interleukin 2- and interferon alpha-induced CD69 antigen expression in human resting NK cells and CD3+, CD4-, CD8- lymphocytes.

The effect of rIL-4 on CD69 antigen expression induced by rIL-2 or by rINF-alpha on human resting NK cells and CD3+, CD4-, CD8- T lymphocytes has been investigated. rIL-4 drastically inhibited CD69 antigen expression induced by rIL-2 in both cell types. In contrast, rIL-4 did not alter rINF-alpha-induced CD69 antigen expression. Consistent results were obtained evaluating the cytolytic activity of NK cells against the Raji target cell line: rINF-alpha-induced lytic activity was not inhibited by rIL-4, while rIL-2-induced lytic activity was drastically inhibited. Proliferative activity of NK cells induced by rIL-2, in contrast, was only slightly reduced by rIL-4. rIL-4 did not alter the expression of the beta chain of IL-2 receptor, evaluated in NK cells by indirect immunofluorescence. Expression of the alpha chain of IL-2 receptor could not be detected in NK cells by indirect immunofluorescence. It can therefore be suggested that the selective inhibitory effect of rIL-4 on rIL-2-induced activation of NK cells is not mediated by downregulation of alpha and beta chains of IL-2 receptor.

Increase of poly(ADP-ribose) polymerase mRNA levels during TPA-induced differentiation of human lymphocytes.

The non-mitogenic stimulation of human peripheral blood mononuclear cells (PBMC) with low concentrations of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) caused a progressive increase in the percent fraction of the cells that were positive for the early activating antigen CD69. At the same time, it caused a progressive increase in the steady-state levels of poly(ADP-ribose) polymerase (pADPRP) transcripts. A further increase in TPA concentration, while inducing the maximal expression of the levels of CD69 activating surface antigen, both in the presence or in the absence of proliferative activity, did not evoke any additional hightening of pADPRP mRNA levels. Time course of PBMC stimulation with a non-mitogenic dose of TPA showed an early increase in the accumulation of pADPRP mRNA, which changed at 8-16 h, and remained high for several days thereafter. On the basis of these data, we suggest that the increase in pADPRP mRNA may be associated with the commitment of human lymphocytes from a quiescent (G0) to an activated (G1) state.

Constitutive expression of CD69 in interspecies T-cell hybrids and locus assignment to human chromosome 12.

In this study we describe the generation and characterization of interspecies somatic cell hybrids between human activated mature T cells and mouse BW5147 thymoma cells. A preferential segregation of human chromosomes was observed in the hybrids. Phenotypic analysis of two hybrids and their clones demonstrated coexpression of CD4 and CD69 antigens in the same cells. Segregation analysis of an informative family of hybrids followed by molecular and karyotype studies clearly demonstrated that the locus encoding CD69 antigen mapped to human chromosome 12. Although the expression of CD69 antigen is an early event after T-lymphocyte activation and rapidly declines in absence of exogenous stimuli, in the hybrids described in this study the expression was constitutive, similarly to what was previously found in early thymocyte precursors and mature thymocytes. In this respect it was important to note that the behavior of the hybrids in culture strongly suggested a dominant influence of the thymus-derived mouse tumor cell genome in controlling the constitutive expression of human CD69. These hybrids may thus provide a system to study the genetic and molecular mechanisms controlling the expression and function of this activation antigen.

Structural analysis of the CD69 early activation antigen by two monoclonal antibodies directed to different epitopes.

The biochemical structure of CD69 early activation antigen has been characterized by means of two newly isolated mAb, namely C1.18 and E16.5. Upon analysis by SDS-PAGE, C1.18-reactive molecules immunoprecipitated from 125I-surface labeled PMA activated PBL consisted of a 32 + 32 kD dimer, a 32 + 26 kD dimer, a 26 + 26 kD dimer and a 21 + 21 kD dimer. E16.5-reactive molecules consisted of a 26 + 26 kD dimer and a 21 + 21 kD dimer. Cross absorption experiments showed that E16.5 mAb reacts with an epitope of the CD69 molecule distinct from the one recognized by C1.18 mAb and present only on a subpopulation of the CD69 molecular pool. The patterns of migration of C1.18- and E16.5-reactive molecules in two-dimensional gel-electrophoresis, under reducing conditions before and after treatment with Endoglycosidase F enzyme suggest that the two mAb recognize the same glycoprotein structure, but in two distinct glycosylation forms, both expressed on the cell surface membrane. Finally, p32, p26 and p21 of CD69 complex obtained from three distinct normal donors did not show appreciable structural polymorphism, by two-dimensional peptide mapping, not only among single subunits within the same individual, but also among homologous subunits in distinct individuals. Further, it was found that CD69 complex is expressed at the cell surface of resting PBL, although at a very reduced level in comparison to PMA activated cells. C1.18 and E16.5 mAb induced comparable cell proliferation and IL-2 production in PBL in the presence of PMA. C1.18 mAb increased intracellular free calcium concn in PMA activated PBL after cross-linking with goat anti mouse Ig, while the effect induced by E16.5 mAb after cross-linking was consistently lower. Finally, it was found that Sepharose-linked C1.18 mAb, in the presence of rIL-2 or PMA, did not induce TNF release from 6 NK cell clones.

Human recombinant interleukin-4 inhibits lymphokine-activated killer activity of sheep erythrocyte rosette-forming (E+) and -non-forming (E-) human lymphocytes.

The effect of human recombinant interleukin-4 (rIL-4) on lymphokine-activated killer activity (LAK) of peripheral blood lymphocytes (PBL) as well as sheep erythrocyte rosette-forming (E+) and -non-forming (E-) lymphocytes stimulated by human recombinant interleukin-2 (rIL-2) has been investigated. rIL-4 drastically inhibited LAK activity of PBL cultured for 18 hr and for 5 days in the presence of rIL-2. Distribution of T, B and IgG Fc-receptor-bearing lymphocytes, as assessed by immunofluorescence and flow cytometry, was no different at the end of the culture in the presence of rIL-2 plus rIL-4 than with rIL-2 alone. LAK activity of E+ and E- lymphocytes was similarly inhibited. Finally, rIL-4 did not affect the natural killer (NK) activity of rIL-2-activated PBL as assessed by the capacity of these cells to kill the appropriate NK target.

Heterogeneity of lymphokine-activated killer (LAK) populations at the clonal level: both NK and CD3+, CD4-, CD8- clones efficiently mediate tumor cell killing.

To investigate at the clonal level the phenotypic and functional properties of interleukin 2 (IL-2) activated killer cells (LAK), recombinant IL-2 activated peripheral blood lymphocytes were cultured under limiting conditions. Among 56 clones that lysed P815 in the presence of phytohemagglutinin (PHA) (22% of total proliferating microcultures) 36 clones lysed also the natural killer (NK)-sensitive K562 and the NK-resistant Hu126 glioma cell lines and one clone lysed only the K562 cell line. Several LAK clones were further assayed for both phenotype and functional activity. Of 22 clones, 10 were CD3-, CD4-, CD8-, and expressed the CD16 marker of NK cells; only one clone had the conventional phenotype of cytolytic T cells (CD3+, CD4-, CD8+), while 11 clones were CD3+, CD4-, CD8- and did not express alpha/beta heterodimer of T-cell antigen receptor as identified by WT31 monoclonal antibody. Only one of the latter clones was CD16+. Endogenous production of IL-2 after stimulation with PHA and phorbol myristate acetate was positive in 3/9 CD3- and in 8/8 CD3+, CD4-, CD8- clones. CD3- mediated strong antibody-dependent cellular cytotoxicity, a function exerted also by some CD3+, CD4-, CD8- T-cell clones to a lower extent. CD3+, CD4-, CD8- T-cell clones lysed different major histocompatibility complex unrelated tumor targets; moreover, this lytic activity seems to be CD3 dependent.