CD3-stimulated Jurkat T cells mediate IL-1 beta production in monocytic THP-1 cells. Role of LFA-1 molecule and participation of CD69 T cell antigen.
Serge N. ManiéJoanna KubarMartine LimouseBernard FerruàMichel TicchioniJ P BreittmayerJean‐François PeyronL. SchaffarBernard Rossi
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In this study we investigated the T cell signals required for monocyte activation. We used an in vitro co-culture system involving two human cell lines: Jurkat T cells and THP-1 monocytes. Monocyte activation was monitored by measuring IL-1 beta production, whereas IL-2 secretion reflected Jurkat activation. We showed that CD-3 -stimulated Jurkat cells delivered an IL-1-inductive signal to THP-1 cells through a cellular contact which was independent of THP-1 Fc receptors cross-linking. Stimulation of IL-1 beta production did not appear to require lymphokine secretion by T cell since a lymphokine defective mutant of Jurkat cell was able to deliver the stimulatory signal. The LFA-1 molecule was clearly shown to participate in the cooperation process, but its role was likely to be restricted to mediating initial adhesive interaction rather than to transducing the IL-1 -inductive signal. Interestingly, the co-culture stimulated by (Fab')2 fragments of CD3 mAb displayed an enhanced IL-1 beta production without any increase of IL-2 secretion. This result indicated that Jurkat cells could stimulate THP-1 cells even when they were only partially activated. The kinetics and conditions of IL-1 beta production called our attention to the early T cell activation antigen CD69. We then showed that CD69 mAb interfered with transmission of the IL-1 inductive signal (40-50% inhibition of IL-1 production). Our results are suggestive of a new role for CD69 molecule intervening in the T lymphocyte-dependent monocyte activation process.Keywords:
Jurkat cells
Monocyte
THP1 cell line
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Previous studies on T cell activation via CD43 antigen stimulation were limited to the use of L10, a monoclonal antibody (mAb) recognizing a sialic acid-independent epitope on the CD43 molecule. Here we study the CD43 mAb MEM-59, which recognizes a neuraminidase-sensitive epitope on the CD43 molecule, for its ability to activate T lymphocytes. The antibody by itself is able to stimulate proliferation of peripheral blood mononuclear cells (PBMC) in a monocyte-dependent fashion, and to act synergistically with the mitogen phorbol 12-myristate 13-acetate. It is demonstrated that the monocyte dependence of MEM-59-induced proliferation of peripheral blood lymphocytes (PBL) cannot be attributed to cross-linking via Fc receptors on monocytes alone: F(ab')2 fragments of MEM-59 are at least as effective as intact IgG in the induction of PBMC proliferation. The effects of MEM-59 reported here are distinct in important ways from those reported for L10. Our proliferation data are extended by the observation that MEM-59 mAb induces mobilization of intracellular Ca2+ in PBMC and in the T cell line Jurkat, while the CD3/TcR-negative Jurkat derived-mutant J.TR3-T3.5 exhibits defective signaling compared to the parent cell line. Moreover, CD3 and CD43 are shown to be present jointly in a large complex in a mild detergent lysate of the T cell line HPB-ALL. These data indicate a physical and functional association between CD3/TcR and CD43 pathways, suggesting a role for CD43 as a co-stimulatory molecule in CD3/TcR signaling, especially in T cell-antigen-presenting cell interactions.
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We have investigated the characteristics of IL2R alpha gene induction in untransformed murine T cells. Induction of IL2R alpha mRNA by TCR/CD3 ligands in a murine T cell clone and in short-term splenic T cell cultures was inhibited by protein synthesis inhibitors and by CsA. This result was contrary to previous observations in JURKAT T leukemia cells and human peripheral blood T cells, suggesting a difference in the mechanisms of IL2R alpha gene induction in these different cell types. The CsA sensitivity of IL2R alpha mRNA induction represented a direct effect on the TCR/CD3 response, and was not due to CsA-sensitive release of the lymphokines IL2 or tumour necrosis factor alpha (TNF alpha) and consequent lymphokine-mediated induction of IL2R alpha mRNA. The NF-kappa B site of the IL2R alpha promoter was essential for gene induction through the TCR/CD3 complex, and the induction of reporter plasmids containing multimers of this site was significantly inhibited by CsA. Northern blotting analysis indicated that while the p65 subunit of NF-kappa B was constitutively expressed and not appreciably induced upon T cell activation, mRNA for the p105 precursor of p50 NF-kappa B was induced in response to TCR/CD3 stimulation and this induction was sensitive to CsA. Electrophoretic mobility shift assays and antiserum against the p50 subunit of NF-kappa B indicated that p50 was a component of the inducible nuclear complex that bound to the IL2R alpha kappa B site. Appearance of the kB-binding proteins was insensitive to CsA at early times after activation (approximately 15 min), but was partially sensitive to CsA at later times. Based on these results, we propose that the NF-kappa B site of the IL2R alpha promoter mediates at least part of the CsA sensitivity of IL2R alpha gene induction in untransformed T cells, possibly because de novo synthesis of p105 NF-kappa B is required for sustained IL2R alpha expression.
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CD3 proteins may have redundant as well as specific contributions to the intracellular propagation and final effector responses of TCR-mediated signals at different checkpoints during T cell differentiation. We report here on the participation of CD3 gamma in the activation and effector function of human mature T lymphocytes at the antigen recognition checkpoint. Following TCR-CD3 engagement of human CD3 gamma-deficient T cell lines, and despite their lower TCR-CD3 surface levels compared to normal controls, mature T cell responses such as protein tyrosine phosphorylation and the regulation of expression of several cell surface molecules, including the TCR-CD3 itself, were either normal or only slightly affected. In contrast, other physiological responses like the specific adhesion and concomitant cell polarization on ICAM-1-coated dishes were selectively defective, and activation-induced cell death was increased. Our data indicate that CD3 gamma contributes essential specialized signaling functions to certain mature T cell responses. Failure to generate appropriate interactions may abort cytoskeleton reorganization and initiate an apoptotic response.
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Abstract Immunotoxins (ITs) were prepared by covalently coupling ricin to monoclonal antibodies (MAbs) directed against: (a) 2 different epitopes of the T‐cell receptor (TcR) expressed by the Jurkat leukemia T‐cell line (JTi 2 and JTi 4 MAb), (b) 2 epitopes of the CD3 complex (SpV‐T3b and IID8 MAb), (c) the CD2 and the CD8 cell‐surface molecules. Conjugates were assayed for their cytotoxic activity by pre‐incubating the Jurkat cell line with different concentrations (10‐250 ng/ml) of each IT for 2 hr at 37°C in the presence of 0.1 M lactose. After washing, cells were cultured for 24 hr and their protein synthesis and proliferative capacities were assessed. Doseresponse experiments indicated that JTi 2 , JTi 4 and anti‐CD3 (IID8) ITs inhibited by >90% the cell line proliferation at 50 ng/ml, a 5‐fold lower concentration than that required to achieve a similar effect when anti‐CD2 and anti‐CD3 (SpVT3b) were used. After 4 hr of culture subsequent to treatment with JTi 2 or JTi 4 ITs (250 ng/ml), protein synthesis was inhibited (>80%). By limiting dilution analysis (LDA) we estimated that the frequency of proliferating Jurkat cells (1/1.5) was reduced to 1/20, 1/460 and 1/300 after treatment with anti‐CD3 (SpVT3b), JTi 4 and JTi 2 ITs, respectively. Phenotypic analysis of 13 clones derived from JTi 2 IT‐treated Jurkat cells showed that 50% were CD7 + CD3 − JTi − variants. When bone‐marrow mononuclear cells, previously mixed with low concentrations of Jurkat cells, were treated with anti‐JTi ITs, the toxic efficiency estimated by LDA was maintained whereas the growth of CFU‐GM remained unaltered.
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In the rat, monoclonal antibodies (mAbs) against the T-lymphocyte receptor (TCR), CD3 antigen and CD2 antigen are mitogenic for T lymphocytes. Antibodies against CD43 (leukosialin) and CD5 are not, but can enhance the effects of other stimuli. Activation of a T-lymphocyte cell line in terms of triggering an increase in free cytoplasmic Ca2+ [( Ca2+]i) was mediated by mAbs against Class 1 MHC and CD4 in addition to the mitogenic mAbs. In the presence of non-activating levels of anti-TCR mAb a strong synergistic signal was seen with anti-CD4, but incubation with anti-CD4 prior to activation with anti-CD3 inhibited the CD3 signal. Stimulation of DNA synthesis in resting T cells via anti-CD2 mAbs required a combination of two non-competitive antibodies and addition of a third anti-CD2 mAb inhibited the activation. The anti-CD2 mAbs were active on all types of mature T cells from peripheral lymphoid organs and the thymus, but cells in the thymus that lacked a TCR could not be activated via CD2. Rat CD2 was transfected into Jurkat cells and cross-linking with anti-rat CD2 mAbs gave an increase in [Ca2+]i similar to that seen with an anti-human CD3 mAb. All three types of anti-rat CD2 mAbs gave a signal including the mAb that inhibited mitogenesis. Signal transduction via CD2 was not seen in L cells and was only weakly observed in TCR-ve Jurkat cells that had been transfected with rat CD2. The cytoplasmic domain of transfected CD2 was shown to be important for the signal transduction since no increase in [Ca2+]i could be triggered in cells expressing mutant CD2 that lacked the cytoplasmic domain. Mutant forms with increasing lengths of cytoplasmic domain showed functions that progressively approached that of the native function.
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The Jurkat E6.1 clone has been extensively used as a powerful tool for the genetic and biochemical dissection of the TCR signaling pathway. More recently, these cells have been exploited in imaging studies to identify key players in immunological synapse (IS) assembly in superantigen-specific conjugates and to track the dynamics of signaling molecules on glass surfaces coated with activating anti-CD3 antibodies. By comparison, Jurkat cells have been used only scantily for imaging on supported lipid bilayers (SLBs) incorporating laterally mobile TCR and integrin ligands, which allow to study synaptic rearrangements of surface molecules and the fine architecture of the mature IS, likely due to limitations in the assembly of immune synapses with well-defined architecture. Here we have explored whether upregulating the low levels of endogenous LFA-1 expression on Jurkat E6.1 cells through transduction with CD11a- and CD18-encoding lentiviruses can improve IS architecture. We show that, while forced LFA-1 expression did not affect TCR recruitment to the IS, E6.1 LFA-1 high cells assembled better structured synapses, with a tighter distribution of signaling-competent TCRs at the center of the IS. LFA-1 upregulation enhanced protein phosphotyrosine signaling on SLBs but not at the IS formed in conjugates with SEE-pulsed APCs, and led to the constitutive formation of an intracellular phosphotyrosine pool co-localizing with endosomal CD3ζ. This was paralleled by an increase in the levels of p-ZAP-70 and p-Erk both under basal conditions and following activation, and in enhanced Ca 2+ mobilization from intracellular stores. The enhancement in early signaling E6.1 LFA-1 high cells did not affect expression of the early activation marker CD69 but led to an increase in IL-2 expression. Our results highlight a new role for LFA-1 in the core architecture of the IS that can be exploited to study the spatiotemporal redistribution of surface receptors on SLBs, thereby extending the potential of E6.1 cells and their derivatives for fine-scale imaging studies.
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In this study we investigated the T cell signals required for monocyte activation. We used an in vitro co-culture system involving two human cell lines: Jurkat T cells and THP-1 monocytes. Monocyte activation was monitored by measuring IL-1 beta production, whereas IL-2 secretion reflected Jurkat activation. We showed that CD-3 -stimulated Jurkat cells delivered an IL-1-inductive signal to THP-1 cells through a cellular contact which was independent of THP-1 Fc receptors cross-linking. Stimulation of IL-1 beta production did not appear to require lymphokine secretion by T cell since a lymphokine defective mutant of Jurkat cell was able to deliver the stimulatory signal. The LFA-1 molecule was clearly shown to participate in the cooperation process, but its role was likely to be restricted to mediating initial adhesive interaction rather than to transducing the IL-1 -inductive signal. Interestingly, the co-culture stimulated by (Fab')2 fragments of CD3 mAb displayed an enhanced IL-1 beta production without any increase of IL-2 secretion. This result indicated that Jurkat cells could stimulate THP-1 cells even when they were only partially activated. The kinetics and conditions of IL-1 beta production called our attention to the early T cell activation antigen CD69. We then showed that CD69 mAb interfered with transmission of the IL-1 inductive signal (40-50% inhibition of IL-1 production). Our results are suggestive of a new role for CD69 molecule intervening in the T lymphocyte-dependent monocyte activation process.
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Monocyte
THP1 cell line
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