ABSTRACT
Human NK cell activity can be augmented in vitro by stimulation with IL-2 or IL-12, both of which also induce the production of IFN-gamma, TNF-alpha, and granulocyte-macrophage CSF by NK cells. For the first time, we demonstrate that freshly purified NK cells stimulated with IL-2 proliferated and produced IL-10 in a dose-dependent manner. IL-10 mRNA expression, as detected by semiquantitative reverse transcription-PCR, reached peak levels at 24 h. IL-10 protein was detectable on day 2 and further increased on days 3 and 6 as measured by ELISA. However, IL-12 alone induced neither substantial proliferation nor detectable IL-10 production by fresh NK cells, but it synergized with IL-2 in inducing IL-10 mRNA expression and protein synthesis. IL-10 production by activated NK cells was confirmed by intracytoplasmic cytokine staining by three-color immunofluorescence of CD16+ and/or CD56+ NK cells with anti-IL-10 antibody. IL-10 production by NK cells was further confirmed in the NK-like cell line, YT, which constitutively expressed IL-10 mRNA and protein. IL-12 alone did not induce NK proliferation, but it inhibited IL-2-induced proliferation. Neutralization of endogenously produced IL-10 with anti-IL-10 antibodies did not overcome the inhibition of IL-2-induced proliferation by IL-12. Together, these results demonstrate that IL-2 and IL-12 synergize to induce IL-10 production by human NK cells and that IL-12 inhibits IL-2 induced NK cell proliferation by an IL-10-independent mechanism.
Subject(s)
Interleukin-10/biosynthesis , Interleukin-12/pharmacology , Interleukin-2/pharmacology , Killer Cells, Natural/drug effects , Cells, Cultured , Flow Cytometry , Humans , Interleukin-10/genetics , Killer Cells, Natural/metabolism , Lymphocyte Activation/drug effects , Polymerase Chain Reaction , RNA, Messenger/analysisABSTRACT
To elucidate mechanisms by which human CD4+ cells mediated cytolytic activity, we studied the expression of cytolytic proteins and the effects of inhibitors and mAbs on T-cell clones. Of seven cytolytic CD4+ clones, three were specific for the HLA-DR17, while four recognized DR18. Anti-HLA-DR mAb and anti-CD4 mAb blocked lysis. In addition, N alpha-p-tosyl-L-lysine chloromethylketone (TLCK), a serine esterase inhibitor, as well as cytochalasin B and monensin, antagonists of secretory pathways, inhibited CD4+ CTLs, whereas the absence of extracellular Ca+2 or the presence of Ca+2 channel blockers partially inhibited cytotoxicity. CD4+ CTLs induced apoptosis of target cell nuclei and membrane damage simultaneously. The CD4+ clones synthesized perforin and granzyme B and expressed the granule-associated protein TIA-1. Our studies indicate that two distinct mechanisms may contribute to cytolysis by CD4+ clones: (1) a Ca+2-dependent mechanism associated with the cytotoxic granules and (2) a Ca+2-insensitive mechanism.
Subject(s)
CD4-Positive T-Lymphocytes/classification , CD4-Positive T-Lymphocytes/immunology , Cytotoxicity, Immunologic , Base Sequence , CD4-Positive T-Lymphocytes/enzymology , Calcium/physiology , Cell Nucleus/immunology , Clone Cells , Cytotoxicity Tests, Immunologic , Cytotoxicity, Immunologic/drug effects , Exocytosis/immunology , Extracellular Space/immunology , Humans , Molecular Sequence Data , Protein Synthesis Inhibitors/pharmacology , T-Lymphocytes, Cytotoxic/immunologyABSTRACT
We have previously demonstrated that human rIL-12 alone can augment the development of cytotoxic activity in stimulated CD8+ T cells. The present study was undertaken to examine the interactions of rIL-7 and rIL-12 on human peripheral blood T cell activation and CTL differentiation. Purified T lymphocytes were pulsed overnight with immobilized alpha-CD3 and then cultured for 3 additional days with IL-7 and/or IL-12. The combination of IL-7 and IL-12 synergistically enhanced the proliferation of either fresh CD3+ T cells or an IL-2-dependent CD4+ T cell line, Kit-225-K6. This synergy was seen on both subsets of T cells; however, CD8+ T cells were usually more responsive to IL-7 and IL-12 at lower concentrations than were CD4+ T cells. Furthermore, these cytokines additively/synergistically augmented the cytotoxic activity of CD8+ T cells. Abs to IL-2 and IL-2R alpha blocked the synergistic effect on proliferation of CD4+ T cells, but had a minimal effect on the synergistic response of the proliferative and cytotoxic activity of CD8+ T cells. Examination of the effects of IL-7 and IL-12 on the expression of IL-12 receptor on T cells revealed an increase in the subunit of IL-12R by IL-7 as determined by flow cytometric analysis. We analyzed the effects on IFN-gamma production by CD8+ T cells and found that IL-7 alone did not induce detectable levels of IFN-gamma production but together with IL-12 it synergistically enhanced the production of IFN-gamma. We also found that IFN-gamma was probably not required for enhanced CTL activity of CD8+ T cells, because Ab to human IFN-gamma did not block additive/synergistic effects of either cytokine. The synergistic stimulatory activity of IL-7 and IL-12 may be of significance in vivo and may provide an alternative mechanism of stimulating T cells for use in immunotherapy.
Subject(s)
Interleukin-12/pharmacology , Interleukin-7/pharmacology , Lymphocyte Activation/drug effects , T-Lymphocytes/drug effects , Antibodies, Monoclonal/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Cells, Cultured , Cytotoxicity Tests, Immunologic , Cytotoxicity, Immunologic/drug effects , Drug Synergism , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Interferon-gamma/biosynthesis , Interleukin-12/immunology , Interleukin-2/physiology , Interleukin-7/immunology , Lymphocyte Activation/immunology , Receptors, Interleukin/biosynthesis , Receptors, Interleukin-12 , Recombinant Proteins/pharmacology , T-Lymphocytes/immunology , Up-Regulation/physiologyABSTRACT
IL4-Pseudomonas exotoxin (IL4-PE4E) is a chimeric molecule in which human IL-4 is genetically fused to the mutated binding domain of Pseudomonas exotoxin. This molecule binds specifically to human IL-4 receptor-bearing cells. IL4-PE4E was extremely cytotoxic to highly purified anti-CD3-activated CD8+ T lymphocytes. The cytotoxic activity of this molecule was dependent on the activation state of CD8+ T cells: 3- and 4-day activated T cells were very susceptible to the cytotoxic activity of IL4-PE4E compared with 0- to 2-day activated cells. PHA-activated lymphocytes and PBL activated in mixed lymphocyte reaction were also highly sensitive to IL4-PE4E. CD16+ and/or CD56+ highly purified NK cells or highly purified, IL-2-activated NK cells were also very sensitive to the cytotoxic effect of IL4-PE4E. IL-2-activated LAK cells had little susceptibility after 1 day but were very sensitive to IL4-PE4E after 3 days. The cytotoxic effects of IL4-PE4E were mediated through a ligand receptor interaction because excess rIL-4 abrogated these effects as did a neutralizing Ab to human IL-4. A chimeric mutant protein that can bind to IL-4 receptors but lacks the ability to inhibit protein synthesis was not cytotoxic to activated lymphocytes. The IL4-PE4E-mediated cytotoxicity of activated T cells correlated with the level of expression of IL-4 receptors on these cells. CD8+ T cells activated for 3 days expressed the highest density of IL-4 receptors compared with 1- or 2-day activated cells. Among two chimeric toxins tested only IL4-PE4E was cytotoxic to 2-day anti-CD3-activated CD8+ T lymphocytes, whereas IL6-PE4E was not active at all. These studies suggest that human IL4 toxin could be a potent agent for the elimination of activated lymphocytes in allograft rejection, some autoimmune diseases, or treatment of lymphomas and leukemias.
Subject(s)
ADP Ribose Transferases , Bacterial Toxins , Exotoxins/chemistry , Interleukin-4/chemistry , Lymphocyte Activation/drug effects , Lymphocyte Subsets/drug effects , Recombinant Fusion Proteins/toxicity , Virulence Factors , CD8 Antigens/analysis , Isoantigens/immunology , Killer Cells, Lymphokine-Activated/drug effects , Killer Cells, Natural/drug effects , Phytohemagglutinins/pharmacology , Receptors, Interleukin-4 , Receptors, Mitogen/metabolism , Structure-Activity Relationship , Pseudomonas aeruginosa Exotoxin AABSTRACT
We have studied the effects of human rIL-12 on the proliferation and generation of cytotoxic activity in human CTL precursors. Purified human blood CD8+ T lymphocytes were stimulated overnight with immobilized alpha-CD3 and cultured 3 to 4 additional days under various conditions. The addition of IL-12 resulted in a marked (10- to 20-fold), dose-dependent, augmentation of cytotoxicity per cell with a smaller (2-fold) increase in cell number. IL-12 augmentation of proliferation and cytotoxicity of CD8+ T cells was not inhibited by a mAb to the p55 subunit of the IL-2 receptor (alpha-Tac) at a concentration sufficient to block the activity of exogenously added IL-2, indicating that the activity of IL-12 did not require IL-2. Addition of IL-12 at the time of alpha-CD3 activation or 1 day later was highly effective at augmenting cytotoxicity, whereas delayed addition of IL-12 (day 2 or 3) resulted in a smaller increase in CTL activity with no increase in cell number. IL-12 at all doses tested synergized with low dose IL-2 in inducing the proliferation and differentiation of CD8+ T cells. The synergistic effect was not blocked by adding neutralizing serum to IFN-gamma. In contrast to this synergistic effect, IL-12 significantly inhibited the proliferation observed in the presence of higher concentrations of IL-2 (4,500 and 13,500 pg/ml). An inhibitory effect of IL-12 was also observed when IL-12 was added to CD8+ T lymphocytes 3 days subsequent to activation with alpha-CD3 and IL-2. This broad set of potent effects of IL-12 on CD8+ T cell responses suggests that IL-12 may play an important immunoregulatory role on CTL development in vivo and may be a useful tool for manipulating this process in vivo for investigational and immunotherapeutic purposes.
Subject(s)
CD8 Antigens/analysis , Cytotoxicity, Immunologic/drug effects , Interleukins/pharmacology , T-Lymphocytes, Cytotoxic/immunology , Cells, Cultured , Drug Synergism , Humans , Interleukin-12 , Interleukin-2/pharmacology , Interleukin-4/pharmacology , Lymphocyte Activation/drug effects , Recombinant Proteins/pharmacology , T-Lymphocytes, Cytotoxic/drug effectsABSTRACT
Varying the concentration of anti-CD3 immobilized on Sepharose beads allowed us to study both the inhibitory and the stimulatory effects of IL-4 on purified T cells and contrast IL-4 versus IL-2-driven T-cell proliferative responses. In the presence of low density immobilized anti-CD3, IL-4 was unable to stimulate purified T cells and was inhibitory to IL-2-driven T-cell responses. The inhibitory effects of IL-4 were enhanced by preincubation of T cells with IL-4 prior to stimulation. In contrast, the inhibitory effects of IL-4 could be avoided by delaying the addition of IL-4 until Days 3-5 of culture or they could be reversed by the addition of IL-1. In the presence of high-density anti-CD3, IL-4 elicited an IL-2-independent proliferative response by purified T cells or sorted CD8+ cells. Comparison of IL-4-driven versus IL-2-driven T-cell responses demonstrated that IL-2 was able to upregulate mRNA for IL-4 receptors and interferon-gamma, while IL-4 had minimal effects on upregulating mRNA for either the p55 or the p75 IL-2 receptor subunits or interferon-gamma. The timing of the presence of IL-4, the state of T-cell activation, and the nature and strength of the stimulatory signal influenced the regulatory effect of IL-4 on the immune response.