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Reconstitution of deficient T cell receptor ζ chain restores T cell signaling and augments T cell receptor/CD3–induced interleukin-2 production in patients with systemic lupus erythematosus†
Article first published online: 2 JUL 2003
Copyright © 2003 by the American College of Rheumatology
Arthritis & Rheumatism
Volume 48, Issue 7, pages 1948–1955, July 2003
How to Cite
Nambiar, M. P., Fisher, C. U., Warke, V. G., Krishnan, S., Mitchell, J. P., Delaney, N. and Tsokos, G. C. (2003), Reconstitution of deficient T cell receptor ζ chain restores T cell signaling and augments T cell receptor/CD3–induced interleukin-2 production in patients with systemic lupus erythematosus. Arthritis & Rheumatism, 48: 1948–1955. doi: 10.1002/art.11072
- Issue published online: 2 JUL 2003
- Article first published online: 2 JUL 2003
- Manuscript Accepted: 24 MAR 2003
- Manuscript Received: 27 JAN 2003
- NIH. Grant Number: R01-AI-42269
T cells from a majority of patients with systemic lupus erythematosus (SLE) display antigen receptor–mediated signaling aberrations associated with defective T cell receptor (TCR) ζ chain, a subunit of the TCR/CD3 complex. This study was undertaken to explore the possibility that forced expression of TCR ζ chain may reverse the known signaling abnormalities and defective interleukin-2 (IL-2) production in SLE T cells.
Freshly isolated SLE T cells were transfected with TCR ζ chain construct in a eukaryotic expression vector at high efficiency, by a recently developed nucleoporation technique. Restoration of TCR/CD3-mediated signaling was studied in the ζ chain–transfected cells.
In SLE T cells transfected with TCR ζ chain, surface expression of TCR chain was increased and the TCR/CD3-induced increased free intracytoplasmic calcium concentration response was normalized, as was hyperphosphorylation of cellular substrates. Simultaneously, the previously noted increased expression of the Fc receptor γ chain was diminished in SLE T cells transfected with the ζ chain expression vector, and the surface membrane clusters of cell signaling molecules were redistributed to a more continuous pattern. TCR ζ chain replacement also augmented the expression of diminished TCR/CD3-mediated IL-2 production in SLE T cells, associated with increased expression of the p65 subunit of nuclear factor κB in the nuclear fractions of these T cells.
These results suggest that reconstitution of deficient TCR ζ chain can reverse the TCR/CD3-mediated signaling abnormalities as well as the defective IL-2 production in T cells of patients with SLE.
It is well recognized that T cells from patients with systemic lupus erythematosus (SLE) display a number of signaling abnormalities (1). Many of the identified molecular aberrations explain certain established cell and cytokine defects, whereas the mechanisms of other defects have not yet been elucidated. Our group and others have demonstrated that the expression of the ζ subunit of the T cell receptor (TCR) is decreased in a majority of SLE patients (2–4) and that this defect persists over time and is independent of disease activity (5).
Despite the decreased expression of the TCR ζ chain in SLE T cells, crosslinking of the TCR/CD3 complex leads to increased free intracytoplasmic calcium concentration ([Ca2+]i) response (6) and protein tyrosine phosphorylation (2, 4). These events appear to occur because the Fc receptor (FcR) γ chain becomes a functional part of the TCR/CD3 complex (7). In support of this view is the observation that forced expression of FcRγ renders normal T cells hyperresponsive in a manner similar to that of SLE T cells (8).
Interleukin-2 (IL-2) production in SLE T cells is decreased because of limited transcriptional activity of the IL-2 promoter due to increased expression of the repressor cAMP response element modulator (CREM) (9) and decreased expression of the p65 chain of nuclear factor κB (NF-κB), which leads to decreased NF-κB activity (10). Our group recently demonstrated that substitution of the defective p65 subunit of NF-κB enhanced TCR/CD3-mediated IL-2 production in SLE T cells (11).
In the present study, we investigated how forced replacement of the TCR ζ chain in SLE T cells would affect the known aberrant TCR/CD3-initiated signaling and gene expression events. Our findings indicate that ζ chain transfection in SLE T cells can indeed correct a number of aberrant events and can restore IL-2 production.
PATIENTS AND METHODS
Patients and controls.
Ten patients who fulfilled the American College of Rheumatology criteria for the diagnosis of SLE (12) were chosen for the study. Nine patients were female and 1 was male. The patients ranged in age from 19 to 80 years. Two were Asian, 5 were African American, and 3 were white. The patients' scores on the SLE Disease Activity Index (13) ranged from 0 (3 patients) to 22 (2 patients). One patient was not receiving any medications, 2 were receiving only hydroxychloroquine, 4 were receiving prednisone and hydroxychloroquine, and 3 were receiving prednisone and cytotoxic drugs. Patients who were receiving prednisone were asked not to take this medication for at least 24 hours before blood was to be drawn. After written informed consent was received, a heparinized peripheral venous blood sample was obtained from each patient. The study protocol was approved by the Health Use Committees of the Uniformed Services University of the Health Sciences, the Walter Reed Army Institute of Research, and Walter Reed Army Medical Center.
The TCR ζ chain monoclonal antibody (mAb) 6B10.2, recognizing amino acids 31–45 of the polypeptide (N-terminal mAb), was purchased from BD PharMingen (San Diego, CA). The C-terminal TCR ζ chain mAb, recognizing amino acids 145–161, and horseradish peroxidase (HRP)–conjugated antiphosphotyrosine mAb 4G10 were purchased from Upstate Biotechnology (Lake Placid, NY).
T lymphocyte isolation.
Peripheral blood mononuclear cells (PBMCs) were obtained by density-gradient centrifugation over Lymphoprep (Nycomed Pharma, Oslo, Norway). Subsequently, T cells were isolated from PBMCs by depletion of non–T cells using a cocktail of hapten-conjugated antibodies and magnetic separation on a MACS column (Miltenyi Biotec, Auburn, CA) as described earlier (7). In all cases, the percentage of T cells in the isolated subpopulation was >98% as determined by anti-CD3 staining and fluorescence analysis with a Coulter counter (Coulter, Hialeah, FL).
Cloning of TCR ζ chain gene in eukaryotic expression vector.
The TCR ζ chain complementary DNA from nucleotides 34–563 without the stop codon was amplified with a high-fidelity polymerase chain reaction (PCR) system. The PCR product was cloned to pCDNA3.1/HIS-TOPO vector (Invitrogen, Carlsbad, CA). Plasmids were isolated from 12 colonies and restriction mapped with Bam HI and Bst XI. The nucleotide sequences of clones with proper orientation were verified by DNA sequencing and used in transfection studies. TCR ζ chain constructs were also made by including the stop codon in the 3′ primer. These constructs express TCR ζ chain without V5 and His6 peptide fusion and are used to rule out the effect of these fusion components.
Transfection by nucleoporation.
T cells (5–20 million) were resuspended in 100 μl nucleofector solution and transferred to a 0.2-cm gap electroporation cuvette. The cells were mixed with 5 μg of plasmid DNA constructs, and nucleoporation was performed on a nucleoporator (Amaxa, Cologne, Germany) using optimized program U-14 at room temperature. Transfected cells were immediately transferred to culture medium and incubated at 37°C in 5% CO2.
Immunoblotting of TCR ζ chain.
T cell activation and antiphosphotyrosine immunoblotting.
T cells were stimulated with 10 μg/ml OKT3 for 0, 1, or 2 minutes at 37°C. The reaction was stopped by the addition of 0.5 ml ice-cold 2× stop buffer (50 mM Tris, 100 mM NaCl, 100 mM NaF, 2 mM Na3VO4, 10 mM EDTA, 10 mM sodium pyrophosphate, 2 mM phenylmethylsulfonyl fluoride, 20 μg/ml leupeptin, and 20 μg/ml aprotinin). Cells were pelleted and lysed in 1% Nonidet P40 buffer (Sigma, St. Louis, MO) as described previously (14). The lysate proteins (20 μg/lane) were analyzed by sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis, immunoblotted with HRP-conjugated antiphosphotyrosine mAb 4G10 (1:1,500), and detected using an enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ).
T cells were suspended in 100 μl of RPMI 1640 (Cell culture medium, Roswell Park Memorial Institute, Buffalo, NY) supplemented with 1% fetal bovine serum and adhered onto poly-L-lysine–coated slides by incubating for 1 hour at room temperature. When necessary, cells were activated with anti–IgM CD3 (clone 2Ad2A2; a kind gift from Dr. Ellis Reinhertz, Dana Farber Cancer Institute, Boston, MA) for 2 minutes at 37°C. The reaction was stopped by addition of 3% paraformaldehyde for 15 minutes, and cells were permeabilized with a buffer containing 0.05% saponin and the blocking antibody (human IgG; 1 μg). Cells were stained with anti-CD3ζ (clone C-20) and anti–linker for activation of T cells (anti-LAT; clone FL-233) (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour at room temperature and counterstained with tetramethylrhodamine isothiocyanate–conjugated anti-goat antibody and fluorescein isothiocyanate–conjugated anti-rabbit antibody (Jackson ImmunoResearch, West Grove, PA), respectively, for 30 minutes. Cells were washed, air dried, and mounted using Gel/Mount (Biomeda, Foster City, CA). Coverslips were applied and the edges sealed with clear nail polish. Samples were analyzed with a laser scanning confocal fluorescence microscope (IX70; Olympus, Lake Success, NY) with Lasersharp2000 software (Bio-Rad, Richmond, CA).
Densitometry and statistical analysis.
Densitometric analysis of the autoradiograms was performed with the software program GelPro (Media Cybernetics, Silver Spring, MD). Statistical analysis of the data was done by paired t-test using the software MINITAB, version 13 (Minitab, State College, PA). P values less than or equal to 0.05 were considered significant.
Transfection and expression of TCR ζ chain in SLE T lymphocytes.
We performed experiments to determine the efficiency of a newly developed nucleoporation protocol for transfection of SLE T cells. Plasmid pIRES containing enhanced green fluorescent protein (GFP) was used as a positive control, and a vector containing β-galactosidase gene was used as a negative control. The data showed that nucleoporation reproducibly results in 70–75% transfection efficiency of GFP in SLE T cells after 18 hours, which is comparable with that of normal T cells (Figure 1A). SLE T cells were then transfected by nucleoporation with TCR ζ chain construct placed in the expression vector pCDNA3.1/HIS-TOPO. After 18 hours, the cells were lysed and the level of TCR ζ chain was measured by immunoblotting with a C-terminal–specific antibody that recognizes the unmodified 16-kd, phosphorylated p21, and 23-kd forms and major ubiquitinated forms of the ζ chain (14). Immunoblotting results revealed high expression levels of all 3 major forms of the ζ chain in TCR ζ chain–transfected SLE T cells (Figure 1B). Under these conditions, the expression of CD3ε or β-actin remained similar in all samples.
Increased surface expression of TCR/CD3 complex in TCR ζ chain–transfected SLE T cells.
Surface levels of TCR/CD3 are decreased in SLE T cells (15). Because TCR ζ chain is critical for the assembly, transport, and surface expression of TCR/CD3 complex (16), we estimated the effect of transfection of TCR ζ chain on the levels of surface TCR/CD3 complex, by fluorescence analysis. As shown in Figure 2, the levels of TCR/CD3 complex were significantly increased in TCR ζ chain–transfected SLE T cells. To determine whether replacement of ζ chain increased the levels of CD3 ε chain or merely facilitated its transfer to the surface membrane, we determined the levels of CD3ε in permeabilized T cells. The data indicate that increases in ζ chain expression facilitate assembly and surface expression of the TCR/CD3 complex, rather than increasing the total levels of CD3 ε chain (Figure 2).
Reconstitution of TCR ζ chain expression reverses the TCR/CD3-mediated increased cytosolic protein tyrosine phosphorylation and [Ca2+]i response in SLE T cells.
One of the hallmarks of SLE T cell signaling abnormalities is the increased TCR/CD3-mediated tyrosine phosphorylation of cellular protein substrates (2) and increased [Ca2+]i response. The kinetics of phosphorylation are also altered in SLE T cells, with peak phosphorylation at 1 minute, in contrast to 2 minutes in normal T cells (2). We examined the tyrosine phosphorylation of cellular protein substrates in TCR ζ chain–transfected SLE T cells after activation for 1 minute with OKT3 antibody. As shown in Figure 3A, TCR ζ chain transfection restored the normal pattern and kinetics of tyrosine phosphorylation of cellular substrates. Similarly, transfection of SLE T cells with the ζ chain construct corrected the heightened calcium response in SLE T cells (Figure 3B).
TCR ζ chain transfection down-regulates FcRγ expression in SLE T cells.
We have shown that SLE T cells, unlike normal T cells, express FcR γ chain, which replaces the deficient TCR ζ chain and participates in T cell signaling events (7). We investigated whether reconstitution of the TCR ζ chain blocked the expression of FcR γ chain in SLE T cells. Indeed, transfection of SLE T cells with TCR ζ chain significantly reduced the expression of FcR γ chain (Figure 4A). Decreased FcRγ protein expression was due to decreased expression of FcRγ messenger RNA (Figure 4B). These data indicate that FcR γ chain is expressed only when the ζ chain is absent or decreased.
TCR ζ chain reconstitution dissolves the clustered membrane distribution of the TCR ζ chain in SLE T cells.
We have recently demonstrated that the residual TCR ζ chain in SLE T cells is distributed in clusters on the surface membrane (14). These ζ chain clusters colocalize with lipid raft markers, suggesting that they represent preaggregated lipid rafts containing signaling molecules. Therefore, we determined the membrane distribution of the ζ chain in ζ chain–transfected and control SLE T cells, by confocal microscopy. After 18 hours of transfection, the cells were mildly fixed, permeabilized, and double-labeled with anti–ζ chain or with an antibody against the lipid raft marker LAT. The confocal microscopy findings showed that transfection with the TCR ζ chain reversed the preformed membrane clusters of the residual TCR ζ chain in SLE T cells, and the ζ chain appeared in a uniform ring pattern (Figure 5). Staining for the lipid raft marker LAT confirmed that preaggregated rafts were reversed. Interestingly, transfection with TCR ζ chain also reversed the faster kinetics of CD3 capping in SLE T cells (Figure 5). Taken together, these findings suggest that reconstitution of the TCR ζ chain normalizes the abnormal morphologic distribution of signaling molecules and membrane dynamics in SLE T cells.
TCR ζ chain transfection augments the production of TCR/CD3-induced IL-2 in SLE T cells.
SLE T cells produce reduced levels of IL-2 upon activation. Decreased expression of the p65 NF-κB chain (10) has been considered to be responsible for the decreased transcriptional activity of the IL-2 promoter. To investigate whether nucleoporation with TCR ζ chain augmented the decreased production of IL-2 in SLE T cells, we measured IL-2 levels in the supernatants of ζ chain–nucleoporated SLE T cells activated with anti-CD3 and anti-CD28 mAb. As shown in Figure 6, the amount of IL-2 secretion was significantly increased in ζ chain–replenished SLE T cells. These results suggest that substitution of TCR ζ chain restores the synthesis of IL-2 in SLE T cells.
To determine whether the increased production of IL-2 was associated with restoration of the decreased expression of the p65 chain of NF-κB, we immunoblotted nuclear fraction proteins from ζ chain–transfected T cells with anti-p65 antibody. As seen in Figure 7, the level of expression of p65 NF-κB in the nuclear extracts was increased by 3-fold in SLE T cells nucleoporated with TCR ζ chain. A similar increase in the level of expression of NF-κB was found in the nuclear fraction of ζ chain–transfected SLE T cells activated with OKT3 antibody (data not shown).
The results presented herein demonstrate that reconstitution of deficient TCR ζ chain in SLE T cells partially restores T cell signaling and function. TCR ζ chain replacement increased the surface expression of the TCR/CD3 complex, normalized the TCR/CD3-mediated phosphorylation of cellular protein substrates and the [Ca2+]i response, and augmented IL-2 production. With these findings, we have shown that low levels of TCR ζ chain play a substantial role in orchestrating T cell signaling abnormalities and T cell lymphokine production, and that maintaining the expression levels of the TCR ζ chain is a vital component for the prevention of signaling aberrations.
Previously, Khan et al (17) showed that bypassing a block in the β regulatory isoform of protein kinase A type I enhances TCR/CD3-mediated IL-2 production. Investigators at our laboratory have demonstrated that reconstitution of p65 NF-κB restores CD3-mediated IL-2 production in SLE T cells (11), as does the elimination of the transcription repressor CREM using an antisense CREM plasmid (18). In those approaches, though, a suboptimal dose of phytohemagglutinin A was used to condition the cells for gene transfer, and this process may have altered cell physiology. The nucleoporation-based transfection technique used in the experiments described herein does not require any cell conditioning, and the transfected gene product is expressed as early as 4 hours (results not shown). In addition, as shown in Figure 1, the transfection efficiency of SLE T cells is high.
We reported previously that the residual ζ chain in SLE T cells can be found in surface membrane clusters along with LAT (14). It appears that the remaining ζ chain localizes in membrane clusters with other signaling molecules to ready the cell to respond to antigen stimulation. In addition, the expressed FcR γ chain contributes to aberrant signaling, as evidenced by the fact that forced expression of FcR γ chain in normal T cells leads to aberrant CD3-mediated signaling reminiscent of that observed in SLE T cells (8). Although increased degradation through the ubiquitin pathway contributes to the decreased expression of TCR ζ chain in SLE (8), it appears that reduced transcription of the ζ chain gene due to decreased production of active DNA-binding Elf-1 (19) is primarily responsible for the reduced production of ζ chain in SLE T cells. We cannot currently explain how replenishment of ζ chain up-regulates the expression of the p65 chain, but it is possible that it may involve phosphorylation of inhibitor of NF-κB, leading to decreased expression of its inhibitory function on NF-κB (20).
Replenishment of missing immunoregulatory molecules by gene therapy in animal models of autoimmune disease has been considered extensively (21). Perfection of the vectors used and of cell transfection protocols may allow the correction of signaling and cytokine defects in human disease. This study shows that effective replacement of a single key signaling molecule in SLE T cells leads not only to correction of signaling defects, but also to restoration of IL-2 production. Decreased IL-2 production in SLE (22) has been associated with increased infection-related morbidity and mortality (23). It is also possible that restoration of ζ chain in tumor-infiltrating cells (24) will effectively increase their tumor cytotoxic ability.