Regulation of Interleukin 2 Receptor Expression by Interleukin 2

The regulatory influence of Interleukin 2 (IL-2) on the expression of IL-2 receptors (IL-2R) was studied using long-term cultured T-cell lines and recombinant IL-2 (r-IL-2). Three T-cell lines with different growth requirements were used as model systems: insulin-specific BK-BI-1.2 cells express IL-2R transiently after antigenic restimulation, ovalbumin-reactive BK-OVA-1R cells express IL-2R permanently, and BK-BI-2.6.C6 cells bear IL-2R constitutively but do not exhibit antigen reactivity. All three T-cell lines exhibited the property of increased IL-2R expression in the presence of r-IL-2, as tested by cytofluorometry employing monoclonal antibody AMT-13 directed at the murine IL-2R. IL-2R density was influenced selectively by r-IL-2, because the level of Thy-1.2 molecules was similar in the presence and absence of r-IL-2. With BK-BI-2.6.C6 cells, r-IL-2 was shown to upregulate high-affinity receptors. Since BK-BI-2.6.C6 and BK-OVA-1R cells were grown in the absence of feeder cells, these data show that r-IL-2 can regulate the expression of its own receptor without the participation of monokines. Results obtained with the T-cell line BK-BI-1.2, representing insulin-specific T cells with transient IL-2R expression, show that the presence of r-IL-2 did not prevent a decline in IL-2R density occurring on day 5 after antigenic stimulus. This indicates that additional mechanisms besides antigen- and IL-2-induced IL-2R upregulation are operative in controlling IL-2R density on the cell surface.     

Activation of Human T Lymphocytes by Phorbol-12,13-Dibutyrate and Ionomycin

The calcium ionophore ionomycin and the phorbol ester phorbol-12,13-dibutyrate (PDBu) are shown to have a synergistic effect upon interleukin 2 (IL-2) production, interleukin 2 receptor expression, and T-lymphocyte proliferation. The proliferative response was inhibited by addition of a monoclonal antibody directed against the IL-2R (Tac antigen) demonstrating that PDBu and ionomycin induce T-cell growth through an IL-2-dependent autocrine pathway. Sequential stimulation with PDBu and ionomycin failed to induce IL-2 production, IL-2R expression, and consequently proliferation of the T cells, indicating that T-cell activation requires simultaneous activation of protein kinase C (PKC) and elevation of cytosolic calcium. Exposure of T cells to both agents for different times resulted in IL-2 production, IL-2R expression, and proliferation in proportion to the duration of incubation with at least 4 h required for maximal T-cell activation. Further, in the presence of PDBu maximal T-cell activation was found to require stimulation with ionomycin for 4 h, indicating that a sustained increase in free cytoplasmic calcium of several hours' duration is essential for T-cell activation. In contrast T cells incubated with ionomycin were induced to produce IL-2 and express IL-2Rs upon brief exposure to PDBu with a 2-h incubation period being sufficient for maximal T-cell activation. Thus transient activation of PKC seems to be sufficient for activation of the IL-2 gene and IL-2R gene. However, maximal T-cell activation requires activation of PKC for at least 2 h.     

CD2-mediated stimulation of the naive CD4+ T-cell subset promotes the development of skin-associated cutaneous lymphocyte antigen-positive memory cells.

Directed migration of lymphocytes from blood into lymph nodes and organ-associated lymphatic tissue, also referred to as homing, is initiated by T-cell adhesion to specialized high endothelial cells of postcapillary vessels. Here, we demonstrate that selective signal transduction pathways specifically modulate the expression of the cutaneous lymphocyte antigen (CLA), the putative skin-homing receptor, during naive to memory transition of CD4+ T cells in vitro. The results show that the expression of CLA is strongly induced by activation via CD2 [T11.1 + T11.2 monoclonal antibodies (mAb)]. Addition of transforming growth factor-beta 1 (TGF-beta 1), interleukin-6 (IL-6), and, to a lesser extent, IL-2 further enhanced the generation of CLA+ T cells, whereas the induction of this antigen was markedly inhibited by IL-4. Periodic restimulation via CD2 and long-term culture of activated cells in the presence of IL-2 and TGF-beta 1 resulted in stable expression of CLA during a culture period of more than 100 days. In contrast, activation of naive CD4+ T cells via CD3, CD28 or by mitogens induced a rapid naive to memory phenotype transition but a much lower percentage of CLA+ T cells showing only weak expression of the antigen. Furthermore, activation of purified CD4+ memory T cells by CD2 strongly induced expression of activation-related antigens CD25 and HLA-DR, but failed to up-regulate CLA expression. Our results show that primary stimulation conditions highly modulate the development of skin-associated T cells and indicate a new functional role for costimulatory adhesion pathways in regulating the expression of molecules associated with T-cell homing.     

In Vitro Activation of Human T Lymphocytes by Haemophilus influenzae Type b Capsular Polysaccharides

Polyribosilribitolphosphate (PRP), the capsular polysaccharide from Haemophilus influenzae type b, is a T-cell-independent type 2 antigen. In vitro culture of adult peripheral blood T cells with 15 micrograms/ml PRP leads to induction of interleukin-2 receptor (IL-2R) expression on up to 10% of T cells. These cells are CD4+ and carry the alpha beta T-cell receptor. PRP, at concentrations above 1-5 micrograms/ml, can also induce in vitro proliferation of both adult and neonatal T cells. We conclude that PRP acts as a human T-cell mitogen. The in vitro proliferative response as well as IL-2R expression was studied in T cells derived from adults after vaccination with native PRP, with PRP conjugated to a carrier protein, or with diphtheria toxoid. Vaccination with conjugated PRP decreased the doses of PRP required for in vitro induction of IL-2R expression and T-cell proliferation. This indicates that vaccination with PRP conjugated to a carrier protein improves the in vitro T-cell response to PRP activation.     

Interaction of IL-1 beta, IL-6 and tumour necrosis factor-alpha (TNF-alpha) in human T cells activated by murine antigens.

We used a mixed leucocyte culture between human T cells and irradiated murine splenocytes which allowed us to distinguish between cytokine production from the responder and stimulator cells by the use of species-specific assays for mRNA up-regulation. Using this model of T cell activation by antigen, we studied the effects of human antigen-presenting cell-derived cytokines IL-1 beta, IL-6 and TNF-alpha on the activation of human T cell subsets. We show in this system that exogenously added IL-1 beta, IL-6 and TNF-alpha induces IL-2 receptor (R) up-regulation and IL-2 production, and proliferation by both CD4+ and CD8+ cells. The addition of IL-1 beta induces IL-6 mRNA, and anti-IL-1 antibodies or an IL-1R antagonist protein completely suppresses IL-6 and TNF-alpha supported proliferation. Similarly, addition of IL-6 or TNF-alpha induces up-regulation of IL-1 beta mRNA. However, anti-IL-6 and anti-IL-6R antibodies only partially block proliferation supported by IL-1 beta. These findings suggest that IL-6 and TNF-alpha will induce IL-2R up-regulation/IL-2 secretion via the induction of IL-1 beta production.     

Mechanism of Calcium Ionophore and Phorbol Ester-Induced T-cell Activation

We examined the role of monocytes in T-cell activation induced by phorbol myristate acetate (PMA) and calcium ionophore ionomycin. Depletion of monocytes from peripheral blood mononuclear cells (PBMC) was associated with the loss of interleukin-2 (IL-2) production, IL-2 receptor (IL-2R) expression and proliferation, in response to either PMA or ionomycin. Addition of monocytes to highly purified T cells resulted in the complete reconstitution of IL-2 production, IL-2R expression and proliferation by PMA-stimulated lymphocytes. Exogenous IL-2, but not interleukin-1 (IL-1), could reconstitute the T-cell responsiveness. Addition of monocytes to highly purified T cells stimulated with ionomycin resulted in partial reconstitution of IL-2 production, IL-2R expression and proliferation. Similarly, the addition of exogenous IL-2 to ionomycin-stimulated T cells only partially reconstituted the response compared with PBMC. These results suggest that monocyte-T-cell interactions contribute to IL-2 production and IL-2R expression and are crucial events for PMA-induced T-cell proliferation. With ionomycin, monocytes play a role, in part, in inducing IL-2 production, IL-2R expression and proliferation. However, IL-2 is not a sufficient signal to induce T-cell proliferative response to ionomycin, suggesting that an IL-2-independent mechanism may exist in ionomycin-induced T-cell proliferation.     

Identification of Th0 cells responding to measles virus

Mechanisms involved in the induction of immunity to measles virus (MV) are not well understood. In the present study, we assessed proliferation, interferon (IFN)-gamma, and interleukin (IL)-4 production of MV-specific T cells after secondary in vitro stimulation of peripheral blood mononuclear cells (PBMCs) from human donors. Such secondary stimulation resulted in responses substantially higher than after primary in vitro exposure. Most study participants produced both IFN-gamma and IL-4 after secondary in vitro stimulation. Patterns of secondary in vitro responses that use genetically disparate antigen-presenting cells were consistent with T-cell recognition restricted to human leukocyte antigen class II molecules. Limiting dilution analyses indicated that precursor frequencies of cytokine secreting and proliferating cells ranged from about 0.001% to 0.1% among fresh PBMCs. Split-well analyses of limiting dilution cultures suggested that virtually all putative T-cell clones produced either IFN-gamma alone or both IFN-gamma and IL-4. Intracytoplasmic flow cytometric analysis of polyclonal MV-specific secondary in vitro responding T cells revealed a similar pattern of cytokine expression. These results suggest that memory T cells responding in vitro to MV generate cells that produce either IFN-gamma alone (and resemble Th1-like cells) or secreted both IFN-gamma and IL-4 (resembling Th0-like cells) in vitro with few cells expressing a Th2-like pattern.     

Antibody to Cryptococcus neoformans capsular glucuronoxylomannan promotes expression of interleukin-12Rbeta2 subunit on human T cells in vitro through effects mediated by antigen-presenting cells

The results reported herein show that T cells responding to encapsulated Cryptococcus neoformans cells had reduced expression of interleukin-12 receptor beta2 (IL-12Rbeta2) in comparison to those responding to non-encapsulated cells. This suggested that encapsulation with glucuronoxylomannan (GXM), the principal constituent of the C. neoformans polysaccharide antiphagocytic capsule, inhibited expression of the IL-12Rbeta2 subunit on T cells responding to cryptococcal antigens. Addition of GXM-binding monoclonal antibody (mAb) overcame this effect by promoting IL-12Rbeta2 expression and by decreasing IL-1R expression on T cells. This effect may be a consequence of mAb-induced changes on antigen-presenting cells (APC) that are closely related to increased phagocytosis. Blocking of phagocytosis with monoiodacetic acid (MIA) precluded up-regulation of B7 expression on APC and was associated with diminished IL-12Rbeta2 expression on T cells. The observed effects on T cells were interpreted as a consequence of increased APC function due to enhanced phagocytosis. These findings suggest a mechanism by which specific antibody can promote the polarization of the cellular immune response towards a Th1-like response and thus contribute to an enhanced cellular immune response against C. neoformans.     

Requirements for the induction of the interleukin-2 receptor complex in a human pre-T-cell line.

Cell line PER-117 is a T-cell receptor negative human T-cell line that can be induced to express a functional interleukin-2 receptor (IL-2R). Recombinant interleukin-1 (IL-1) as well as certain combinations of inducer substances could be shown to stimulate the expression of the p55 (alpha)-chain of the IL-2R in PER-117 cells. The synergistic increases in IL-2R alpha expression were demonstrated at the cell surface as well as at the mRNA level. The results suggested that in PER-117 cells IL-1 appears to induce expression of the alpha-chain by pathways that are different to activation via protein kinase C (PKC), and that drug-induced cyclic AMP (cAMP) activation did not substitute for IL-1. We found that the regulation of mRNA for IL-2R beta (p75) differed significantly from that seen for IL-2R alpha. Moreover, the requirements for IL-2R alpha induction determined for this cell line differ from other human cell lines, which may reflect that there are distinct requirements for activation depending on the stage of differentiation and/or lineage of the cells. The PER-117 cell line provides a unique model to examine further the mechanism leading to induction of a functional IL-2R at an early stage of human T-cell differentiation.     

Immune dysfunction expressed selectively on L3T4+ T cells in the tumor-bearing state

Spleen cells from normal C3H/He or BALB/c mice generate cytotoxic T-lymphocyte (CTL) responses to both trinitrophenyl (TNP)-modified syngeneic cells (TNP-self) and allogeneic cells. In contrast, cells from these strains of mice bearing a syngeneic tumor failed to induce anti-TNP-self-CTL responses, although portions of the same responding cells generated comparable anti-allo-CTL responses to those induced by normal responding cells. Although anti-allo-CTL responses were inducible from only Lyt-2+ T-cell subset of responding cells from normal or tumor-bearing mice, induction of TNP-CTL responses required the participation of L3T4+ T-cell subset as well as Lyt-2+ CTL precursors. In addition, the fact that the addition of concanavalin A-stimulated culture supernatant to cultures of responding cells from tumor-bearing mice resulted in the induction of appreciable anti-TNP-self CTL responses demonstrated the defect of L3T4+ T-cell function in the tumor-bearing state. Such a functional defect was ascribed neither to the loss of L3T4+ T cells nor to the generation of suppressor cells in spleen cells of tumor-bearing mice. It was found on one hand that in vitro stimulation of antigen-presenting cell (APC)-depleted normal responding population with TNP-self prepared from cells of normal or tumor-bearing mice produced comparable anti-TNP CTL responses. On the other hand, APC-depleted responding cells from tumor-bearing mice were unable to induce anti-TNP CTL responses under conditions in which APC-depleted normal responding cells induced an effective TNP-CTL response. These results indicate that selective impairment of L3T4+ T cell-mediated immunity is induced in the tumor-bearing state and that such an impairment is ascribed to the dysfunction of L3T4+ T cells themselves, but not of APC required for the activation of L3T4+ T-cell subset.     

Defective antigen presentation in chronically protein-deprived mice.

Immunodeficiency syndromes associated with protein-energy malnutrition (PEM) have been documented extensively, although to date the mechanism underlying these defects remains uncharacterized. In this study, we have evaluated T, B, and antigen-presenting cell functions of malnourished mice fed a 4% protein diet compared with litter-mate controls fed a 20% protein diet. Spleen cells from malnourished mice presented both soluble foreign protein and allogeneic MHC antigens less efficiently than control mice. However, T cells from malnourished animals demonstrated effective or enhanced specific T-cell activation when stimulated with allogeneic cells, while B cells from protein-deprived animals responded normally in proliferative responses to T-cell driven cognate and non-cognate, as well as mitogen, stimulation. To assess further antigen-presenting cell function, three requirements for successful antigen presentation were evaluated. First, the proliferation of the IL-1-dependent cloned T-cell line D10 demonstrated a slight deficiency in IL-1 production by malnourished splenic antigen-presenting cells, and the addition of saturating amounts of IL-1 to the assay could partially reconstitute function. Second, quantitative cell-sorter analysis revealed minimal deficiencies of spleen-cell Ia expression. Third, antigen-processing function was assayed in vitro by using processed antigen fragments; no improvement in protein-deprived antigen-presenting function resulted. Together, these findings suggest that either decreased Ia glycoprotein expression on a critical subset of antigen-presenting cells (APCs) or a quantitative deficiency in such a subset of cells, or both, underlie the defective antigen-presenting cell function observed in chronic protein deprivation (CPD).     

T-cell activation. I. Evidence for a functional linkage between class I MHC antigens and the Tc-Ti complex

This paper examines the possibility of a functional linkage between class I MHC molecules and the T-cell receptor complex for antigen (T3-Ti). A newly developed anti-CD3 antibody (500A2) was used as an activation signal for EL4 lymphoma cells and allospecific cytotoxic T-cell clones (CTL), and the production of IL-2/IL-2 receptor in EL4 cells and serine esterase in CTL was determined. Anti-CD3 antibody-induced activation of both EL4 and CTL cells was enhanced in the presence of immunologically cross-linked and immobilized anti-H-2 (class I) antibody reactive against the H-2 haplotype of the responding T cells. A number of H-2-negative and H-2-positive EL4 subclones were generated and tested for anti-CD3 antibody-induced IL-2/IL-2 receptor production. Although both H-2-positive and -negative subclones expressed CD3 antigen and produced IL-2 after activation with the phorbol ester TPA, only the H-2-positive cell clones produced IL-2 and expressed IL-2 receptor after anti-CD3 antibody induction. Our results are compatible with the existence of a functional linkage between the class I and the CD3 molecules on the surface of T cells.     

Functional inactivation of Dermatophagoides spp. (house dust mite) reactive human T-cell clones

Staphylococcal enterotoxins are able both to stimulate powerful polyclonal proliferative responses and to induce non-responsiveness of T lymphocytes expressing the appropriate T-cell antigen receptor V beta gene products. T-cell clones representative of the human response to house dust mite were identified that express either V beta 3 or V beta 6 gene products. The specificity of the latter was confirmed by serology. Pre-treatment of cloned V beta 3+ T cells with the Staphylococcus aureus enterotoxins B or C1 rendered them non-responsive to immunogenic challenge with their natural ligand, while retaining responsiveness to exogenous IL-2. Similarly, exposure of the V beta 6+ dust mite reactive T cells to the staphylococcal enterotoxin of the appropriate specificity, SEE, induced specific anergy. The development of non-responsiveness was associated with changes in the T-cell phenotypes. Downregulation of the T-cell receptor, Ti-CD3, was paralleled by enhanced expression of both CD2 and the IL-2 receptor, CD25. Differential co-modulation of CD4 and Ti-CD3 suggested that for some T cells CD4 may form part of the specific antigen recognition structure. Toxicity of the staphylococcal enterotoxins may be removed by chemical modification, thus their ability functionally to inactivate subpopulations of T cells expressing antigen-specific receptors with shared characteristics may be of potential value in the regulation of allergic diseases if the diversity of the T-cell repertoire proves to be limited.     

Responsiveness of T cells to Mutant Major Histocompatibility Complex Class I Antigen

Mechanisms of the activation of T cells responding to major histocompatibility complex (MHC) class I antigen were investigated with special reference to interleukin 1 (IL-1) production from stimulator-type accessory cells. For this purpose, we used mainly fractionated Lyt-2+T cells of C57BL/6 (B6) mice as responder cells and irradiated spleen cells or those deprived of adherent cells of B6.C-H-2bm1 (bm1) mice as stimulator cells. Lyt-2+ T cells of B6 mice proliferated in the presence of irradiated whole spleen cells of bm1 mice but did not to Sephadex G-10 column-passed bm1 spleen cells. The unresponsiveness in the latter case was overcome by the supplement of recombinant IL-1 and/or IL-2 in the culture medium. These interleukins were shown to promote the proliferative response of B6 Lyt-2+ T cells in the presence of stimulator-type T or B cells. Both interleukins also facilitated the generation of cytotoxic T cells from B6 Lyt-2+ cells to H-2Kbm1 antigen in the mixed lymphocyte culture deficient in stimulator-type accessory cells. IL-1 was shown to enhance the expression of IL-2 receptor on the responding Lyt-2+ T cells as assessed by flow cytometry. IL-1 binding to responding T cells were also assayed by means of iodinated IL-1 and was shown to increase significantly on responding Lyt-2+ cells. Overall results indicate that accessory cells might play dual roles in the activation of Lyt-2+ T cells responding to allogeneic MHC class I antigen: direct presentation of the antigen to responder T cells and production of IL-1. Both signals are essentially required for Lyt-2+ T cells responding to allogeneic MHC class I antigen to initiate proliferation and also to differentiate into cytotoxic T cells.     

Bolus injection of aqueous antigen leads to a high density of T-cell-receptor ligand in the spleen, transient T-cell activation and anergy induction.

In vivo anergy can be modelled by administration of soluble peptide to T-cell receptor (TCR) transgenic mice specific for the moth cytochrome c peptide 88-103 (MCCp). Two weeks after initial peptide treatment, T cells were present in normal numbers but were unresponsive to antigen stimulation in vitro. Only bolus injections of peptide, either subcutaneous or intravenous, were effective at inducing tolerance, while slowly released antigen administered via mini-osmotic pump failed to result in anergy. Examination of T cells soon after bolus peptide administration revealed that anergy induction was preceded by a transient hyperactivation of T cells in vivo. Within 2 hr of peptide treatment, interleukin-2 was detectable in the plasma of the transgenic mice. Interestingly, only bolus injections of peptide led to high levels of T-cell activation, while adjuvant emulsified and pump-administered peptide resulted in very low stimulation in vivo. When the dose of bolus-injected peptide used for tolerization was titrated, the extent of anergy induction directly correlated with the intensity of early T-cell activation. Indirect measurements of TCR-ligand density on the surface of antigen-presenting cells following peptide administration revealed that aqueous peptide delivered via bolus injection generated a large number of major histocompatibility complex-peptide complexes, while pump-delivered and adjuvant-emulsified peptide did not. These data suggest that high levels of TCR ligand are required for in vivo T-cell hyperactivation and induction of anergy.     

Poly-guanosine motifs costimulate antigen-reactive CD8 T cells while bacterial CpG-DNA affect T-cell activation via antigen-presenting cell-derived cytokines

Pathogen-derived pattern recognition ligands like lipopolysaccharide (LPS) and bacterial cytidine-guanosine (CpG)-DNA not only activate dendritic cells and macrophages but are also mitogenic for B cells. Less clear are the claimed effects of CpG-DNA on T cells, which range from direct activation, costimulation, or indirect transient activation via antigen-presenting cell (APC)-derived interferon type I (IFN type I). Here we demonstrate that CpG-DNA sequence specifically triggers macrophages to produce IFN type I, interleukin (IL)-12, IL-6 and tumour necrosis factor (TNF), but lacks the ability to directly costimulate T cells. Strikingly, poly-guanosine (poly-G) extensions to CpG-containing oligonucleotides (ODN) abolished the macrophage stimulatory potential yet generated T-cell costimulatory activities. In fact, independently of CpG-motifs, poly-G-ODN displayed the ability to costimulate T cells. Costimulation was operative on CD8 T cells but not CD4 T cells. Poly-G-mediated costimulation resulted in IL-2-driven T-cell proliferation and induced cytolytic T cells. Overall the data imply that poly-G motifs costimulate antigen reactive CD8 T cells, while CpG-DNA motifs fail to do so but may affect T-cell activation via APC derived cytokines such as IFN type I.     

BCG-induced suppressor T cells optimal conditions for in vitro induction and mode of action.

In vitro activation with BCG of T cells from healthy individuals vaccinated with BCG lead to the induction of suppressor cells that suppressed the proliferation of fresh T cells in response to specific antigen. Kinetics of their induction revealed that they became radioresistant by day 8 and persisted up to 18 days of the culture period. Optimal antigen and monocyte concentrations as assessed by proliferation during the induction phase also resulted in maximum suppression. The strongest suppressor activity was observed when suppressor cells were added at an early time of fresh cell activation. IL-1 production from adherent cells in response to BCG was not affected, but, IL-2 production by T-cells was considerably reduced in the presence of suppressor cells. IL-1 containing supernatants and affinity purified IL-1 exogenously added to the culture system did not affect suppression. Whereas, recombinant IL-2 partially abrogated suppression in a dose-dependent manner. Further experiments suggested that suppressor cells might have inhibited BCG induced IL-2 receptor expression on fresh T cells.