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Department of Medicine and Center for Immunology, University of Minnesota Medical School, Minneapolis, MN 55455
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Such a proximal block in signal transduction to the IL-2 gene could be due to the inactivation or degradation of important signaling molecules in the plasma membrane or cytosol of unresponsive T cells. Alternatively, clonal anergy may result from the induction (or overexpression) of molecules capable of inhibiting the transmission of signals from the Ag receptor to the nucleus. To test the first hypothesis, we determined whether fusion of anergic murine Th cells to normal murine or human T cells otherwise capable of secreting IL-2 would be sufficient to result in a complementation of the anergy defect and the restoration of normal IL-2 gene inducibility. We focused our analysis only on the heterokaryons that infrequently formed following exposure to polyethylene glycol (PEG)3 both by using a strategy of species-specific mAbs and RT-PCR to stimulate and detect mRNA for murine IL-2 and by detecting the accumulation of intracellular IL-2 protein in heterokaryons using flow cytometry. Our results indicate that a molecular repressor molecule present within anergic T cells can block the transduction of biochemical signals from the plasma membrane to the IL-2 gene even after cell fusion and heterokaryon formation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Cloned murine CD4+ Th cells (A.E7 and 16B.2) (10, 11) were used as fusion partners with the human T leukemia cell line Jurkat (American Type Culture Collection, Manassas, VA). A murine CD4+ T cell line produced by intermittent in vitro Ag stimulation (three or four cycles) of lymph node cells from the DO11.10 TCR-transgenic mice was also used in the murine/murine heterokaryon experiments (12). T cells in this line uniformly express a TCR that can be recognized by the KJ1–26 anti-clonotypic mAb (13). T cells were grown at 37°C with 5% CO2 in complete medium consisting of a 1/1 mixture of Eagle’s Hanks’ amino acids medium (Biofluids, Rockville, MD) and RPMI 1640 (Celox, Hopkins, MN) containing 10% FCS (HyClone, Logan, UT), 2 mM L-glutamine, penicillin, gentamicin, and 5 x 10-5 M 2-ME. Murine cells were periodically stimulated with irradiated syngeneic splenocytes and Ag and were split 2 days later with 50 U/ml of IL-2. Then they were allowed at least 12 days to return to a resting phenotype before purification on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) and use in experiments.
Clonal anergy induction
Anergy was induced in T cells by incubation overnight at 37°C in flasks coated with 0.5–2 µg/ml of immobilized anti-CD3 mAb in the absence of accessory cells as previously described (8). Thereafter, the cells were removed from the mAb, resuspended in complete medium, and rested for 4–9 days. Before restimulation, viable cells were recovered on a Ficoll-Hypaque density gradient and then stimulated as indicated in the experiment.
Heterokaryon formation
Murine and human T cells were mixed at a ratio of 1.5/1 and suspended in 42% PEG (Life Technologies, Gaithersburg, MD) in PBS for 2 min at room temperature, then gently pelleted by centrifugation at 1000 rpm for 5 min (14). PEG 1000 proved to be the most effective for fusion of these cells. PEG was immediately washed away using serum-free RPMI, and viable cells were recovered on a Ficoll-Hypaque centrifugation gradient. Murine/murine heterokaryons were produced in a similar fashion after mixing of the KJ1–26+ line with either A.E7 or 16B.2 T cells at a 1/1 ratio.
T cell activation
T cells were then cultivated for 5–7 h in complete medium either without stimulation or in the presence of CD3 plus CD28 mAbs as previously described (8). For stimulation of Jurkat cells and the murine/human heterokaryons, purified anti-human CD3 mAb OKT3 (American Type Culture Collection) or 64.1 (a gift from Dr. Peter Lipsky, University of Texas Southwestern Medical Center, Dallas, TX) was immobilized on the culture plate at a concentration of 1–10 µg/ml for 90 min before cultivation of the T cells. The anti-human CD28 mAb 9.3 was in ascites (provided by Dr. Jeff Ledbetter, Oncogen, Seattle, WA) and was added directly to the cultures at a 1/1000 dilution. For stimulation of the murine T cells and all heterokaryons, purified anti-murine CD3 mAb 145-2C11 (15) and anti-murine CD28 mAb 37.51 (3) were coimmobilized at concentrations of 1.0 and 0.3 µg/ml, respectively, except where noted. Alternatively, T cells were activated with the combination of 0.5 µM ionomycin (Iono; Calbiochem, La Jolla, CA) and 10 ng/ml PMA (Calbiochem) to bypass stimulation of the TCR.
RT-PCR
Following stimulation with one or the other mAb pairs, cultures were extracted with RNA-STAT 60 (Tel-Test, Friendswood, TX) according to the manufacturer’s recommendation to recover total cellular RNA. Samples were DNase treated, and 5 µg of RNA was then reverse transcribed using random hexanucleotides (Boehringer Mannheim, Indianapolis, IN) and avian myeloblastosis virus (AMV) reverse transcriptase (Promega, Madison, WI) to produce cDNA. Dilutions of cDNAs were then performed to allow for a semiquantitative estimate of the original murine mRNA steady state levels using the PCR and species-specific PCR primers.
Intron-spanning murine-specific primers used in the study were: IL-2: upstream, 5'-GTCACATTGACACTTGTGCTCC-3'; downstream, 5'-AGTCAAATCCAGAACATGCCG-3' (GenBank accession no. M16760 and M16761); lymphotactin: upstream, 5'-CGGTGGATCCATGAGACTTCTCCTCCTGACTTTCC-3'; downstream, 5'-GGTGAATTCTTACCCAGTCAGGGTTATCGCTGTGC-3' (GenBank accession nos. U28491 and U28493); and HPRT (hypoxanthine phosphoribosyltransferase): upstream, 5'-GTTGGATACAGGCCAGACTTTGTTG-3'; downstream, 5'-GAGGGTAGGCTGGCCTATAGGCT-3' (GenBank accession no. J00423).
Intron-spanning human-specific primers used in the study were: IL-2: upstream, 5'-CACATTAACCTCAACTCCTGCCAC-3'; downstream, 5'-CGTTGATATTGCTGATTAAGTCCCTG-3' (GenBank accession no. X00695); and HPRT: upstream, 5'-GGTCAGGCAGTATAATCCAAAG-3'; downstream, 5'-GTCAATAGGACTCCAGATGTTC-3' (GenBank accession no. M26434).
To enhance primer specificity, annealing temperatures for the initial cycles of the PCR were decreased from 62 to 58°C; then 29 more cycles were conducted with an annealing temperature of 57°C. PCR products were separated by 1% agarose gel electrophoresis and visualized either by ethidium bromide staining within the gel or by Southern blotting to nylon membrane (Magnagraph Micron Separation, Westborough, MA) with detection using human IL-2, murine IL-2, and murine lymphotactin gene cDNA probes. The primers amplified DNAs of the following sizes: murine IL-2, 467 bp; murine lymphotactin, 444 bp; murine HPRT, 432 bp; human IL-2, 344 bp; and human HPRT, 234 bp.
Confocal laser microscopy
The confocal microscopy analysis used PEG-treated cultures incubated for 5 h in complete medium. Viable cells were then attached to coverslips precoated with 1 mg/ml Con A (Pharmacia) by centrifugation. Cells were incubated with anti-human CD45-biotin (PharMingen, San Diego, CA) for 20 min, washed in sorter buffer, and then incubated for 20 min more with anti-murine CD4-FITC (Caltag, South San Francisco, CA) and streptavidin-Cy5 (Jackson ImmunoResearch Laboratories, West Grove, PA). Washed cells were fixed with 2% paraformaldehyde for 20 min and then permeabilized with 1% Triton X-100 (Sigma, St. Louis, MO) for 2 min. Washed cells were exposed to 1 mg/ml RNase A for 10 min at 37°C followed by air-drying. In the final incubation step, ethidium bromide was added at a concentration of 1 µg/ml for 5 min followed by washing of the cells. Coverslips were mounted to slides, and images were collected on a Bio-Rad MRC 1000 confocal laser microscope using COMOS version 6.05.8 software (Bio-Rad Microscience, Herts, U.K.). Grey scale images were electronically collected using 488 nm (murine CD4), 568 nm (ethidium bromide), and 647 nm (human CD45) fluorescence laser excitation; a x60 objective; and a Kalman filter (16 scans). Images were then imported into Adobe Photoshop software (Adobe Systems, San Jose, CA), pseudo-colored (murine CD4 fluorescence emission as green, human CD45 as red, and ethidium as blue), and merged.
Flow cytometry
For the murine/human heterokaryons, PEG-treated cultures were incubated on ice for 30 min with a mixture of anti-human CD45-CyChrome (PharMingen) and anti-murine CD4-PE (PharMingen) in sorter buffer containing 1% FCS and 0.05% sodium azide in PBS. Cell fusion was then analyzed by flow cytometry using a FACScan cytometer and CellQuest software (Becton Dickinson, Mountain View, CA). In the case of murine/murine heterokaryons, A.E7 or 16B.2 T cells were stained with the fluorescence tracking dye carboxyfluorescein diacetate/succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) at a 0.5-nM concentration before fusion. PEG-treated cultures were then stimulated in plates for 2 h with either the combination of CD3 plus CD28 mAbs or with Iono plus PMA. At that time, 10 µg/ml brefeldin A (Sigma) was added to the cultures, and incubation was continued for an additional 5 h. Cultures were harvested and stained with KJ1–26-biotin/streptavidin-CyChrome (PharMingen) followed by fixation with 4% formaldehyde and detection of intracellular IL-2 with anti-IL-2-PE (PharMingen) as previously described (16). CFSE+ KJ1–26+ heterokaryons were electronically gated, and IL-2 accumulation was compared with that of normal KJ1–26+ T cells.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The mRNA harvested from unfused mixtures of murine cloned
CD4+ Th cells and human Jurkat cells contained detectable
transcripts for murine IL-2 only when the cells were
stimulated with an mAb pair specific for murine CD3 and CD28 molecules
(Fig. 1
A, lane 2).
Anti-CD3 and -CD28 mAbs reactive with human molecules, while capable of
inducing the production of human IL-2 mRNA by the Jurkat
cells within the mixed population (Fig. 1A, lane
3), did not stimulate murine IL-2 mRNA detectable by
RT-PCR, confirming the species specificity of both the anti-human
Abs and the PCR primers. In contrast, exposure of mixed cell
populations (murine Th cells plus Jurkat) to PEG resulted in the
appearance of mRNA for murine IL-2 that could be easily
detected after stimulation of the cells with Abs against human CD3 and
CD28 (Fig. 1A, lane 5). This result showed that
functional murine/human heterokaryons do form in the presence of PEG.
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B). Double-labeled
murine/human heterokaryons were similarly detected in PEG-fused
populations using confocal microscopy, and they consistently (in each
of 22 murine/human heterokaryons analyzed) demonstrated the presence of
at least one denser murine T cell nucleus plus one larger human
Jurkat-derived nucleus (Fig. 1
B). Finally, the activation of
a ß-galactosidase reporter gene (regulated by a multimer of the DNA
binding site for the nuclear factor of activated T cells) stably
transfected into Jurkat could be detected by three-color flow cytometry
following stimulation with anti-murine CD3 and CD28 only after
fusion with the mouse cloned T cells (data not shown).
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, lanes 3 and 5). Therefore, sufficient
similarities must exist between the signaling cascades of man and mouse
such that stimulation of human CD3 and CD28 molecules can fully
activate murine gene transcription in the murine/human heterokaryons.
We then took advantage of these similarities to test whether signaling
molecules provided by human Jurkat cells could restore normal signal
transduction to the murine IL-2 gene after fusion with an
anergic murine Th cell. Fusion of anergic murine T cells with Jurkat fails to overcome the anergy defect
Clonal anergy was induced in cloned murine T cells by overnight
exposure to immobilized CD3 mAb to mimic Ag recognition in the absence
of CD28 costimulation, as previously described (8). The T cells were
then allowed to incubate in medium alone for 6 days, in parallel with a
normal control T cell population that had not been exposed to the CD3
mAb. Upon rechallenge with Ag, the CD3-pretreated T cells showed a
substantially reduced IL-2 production and proliferation compared with
the control population (data not shown), consistent with the induction
of clonal anergy. Each group of murine cloned T cells was then mixed
with Jurkat, fused in PEG, and subsequently examined for mRNA synthesis
following stimulation with the CD3 and CD28 mAb pairs (Fig. 2A). Anergic T cells expressed significantly fewer murine
IL-2 mRNA transcripts than control cells upon stimulation
with the anti-murine CD3 and CD28 mAbs (Fig. 2A, compare
lanes 3–7 to lanes 8–11). This was the expected
result, since heterokaryon formation occurred only at low frequency,
and no effect of the Jurkat cells was anticipated on the majority of
the nonfused murine T cells in the culture. Murine
lymphotactin mRNA, on the other hand, was strongly induced
in the anergic group by the anti-murine mAb stimulation as
previously reported (8), again indicating that some signaling pathways
between the plasma membrane and the nucleus remained intact within the
anergic murine Th cells.
Upon stimulation of the PEG-fused anergic group with anti-human CD3
and CD28 mAbs to focus attention solely on the murine/human
heterokaryons in the culture, the defect in murine IL-2 mRNA
induction persisted (Fig. 2A, compare lanes
12 and 13 with lanes 14 and 15).
A correction of the clonal anergy IL-2 mRNA synthesis defect
was never observed in five separate fusion experiments, and murine
IL-2 mRNA inducibility within these anergic heterokaryons
remained reduced by an average of 80 ± 5%. This failure of the
anergic group to synthesize murine IL-2 mRNA in response to
the anti-human mAb pair was not simply the result of a decreased
capacity of anergic murine cells to form a viable heterokaryon with
Jurkat, since murine lymphotactin mRNA could still be
normally induced with the combination of anti-human mAbs (Fig. 2A). Furthermore, anergic T cells were observed to form
heterokaryons with Jurkat with a frequency similar to that in normal
cells (Fig. 2B). Therefore, a defect in signal transduction
to the murine IL-2 gene that exists in anergic T cells could
not be overcome by fusion and mixing of anergic cell contents with
Jurkat.
A dominant-acting repressor molecule in viable anergic T cells
Human IL-2 gene inducibility was also examined
following fusion of Jurkat to the anergic mouse T cells (Fig. 3). Anergic murine/human heterokaryons
demonstrated defective human IL-2 gene transcription
following CD3 and CD28 stimulation with the anti-mouse mAb pair
compared with normal heterokaryons. In five separate experiments, human
IL-2 gene inducibility was reduced 78 ± 9% following
fusion with anergic T cells. Thus, a molecule within the anergic murine
T cell was capable of interrupting the normal cascade of signals from
the plasma membrane to the human IL-2 gene when introduced
into Jurkat T cells.
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A). Even if the human
counterpart of such a putative defective component failed to act in its
place because of a species difference (which is unlikely given the
conservation between man and mouse of the signaling cascades), one
should have expected multiple cross-species interactions within the
signaling cascade of the heterokaryon distal to the murine signaling
block. Instead, the data suggest that the induction of clonal anergy in
CD4+ Th cells results in the expression of a
dominant-acting repressor molecule capable of inhibiting a signal
transduction cascade involving normal components from both species
(Fig. 4B).
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and 6). Nevertheless, fusion of normal
KJ1–26+ T cells with anergic CFSE-stained A.E7 or 16B.2 T
cells yielded a population of CFSE+ KJ1–26+
heterokaryons that was deficient in the production of IL-2 relative to
either normal heterokaryons or unfused KJ1–26+ T cells. In
the case of stimulation with the combination of CD3 and CD28 mAbs, IL-2
production by anergic murine/murine heterokaryons was reduced 48
± 4% compared with that by control heterokaryons (Fig. 5B). In contrast to heterokaryons, IL-2 synthesis by the
majority of KJ1–26+ T cells that remained unfused in the
PEG-treated cultures was not significantly reduced by the presence of
anergic CD4+ T cells.
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Data shown here now document the actions of a repressor molecule in the regulation of endogenous IL-2 gene expression. It should be noted that in these experiments the actions of such a repressor molecule would dominate over the complementation of a missing signaling component, should both have occurred in heterokaryons of anergic T cells. It is possible that both a repressive mechanism and the loss of a signaling component coexist in anergic T cells. Similarly, the observation of a dominant-acting repression does not allow for a determination of the location of the repressor molecule(s) within anergic T cells. Previous biochemical analyses of anergic murine Th cells have indicated a block in signal transduction to p21ras and the mitogen-activated protein kinases (MAPK) (5, 6), resulting in poor induction of Fos and Jun species important to trans-activation at the 5' IL-2 gene enhancer (7, 8). Therefore, these new results could support a model in which a proximal inhibitor molecule blocks the coupling of the Ag receptor to p21ras. Nevertheless, these data may also have resulted from the effects of a nuclear repressor molecule capable of translocating to the heterokaryon’s normal nucleus during the course of the experiment. The zinc finger DNA binding protein called negative regulator of IL-2a (Nil-2a) has the capacity to inhibit trans-activation at the IL-2 gene (26), and overexpression of this repressor molecule has previously been suggested as the basis for clonal anergy in human T cell clones (27). A pair of activating protein-1-like DNA sequences within the 5' IL-2 gene enhancer have also been suggested as the targets of dominant negative regulation in the nucleus of anergic murine T cells (28).
Iono plus PMA activation cannot bypass the effects of the anergy repressor molecule
Previously, we reported that the stimulation of anergic T cells
with the combination of a calcium ionophore and protein kinase
C-activating phorbol ester resulted in a significant, albeit
incomplete, restoration of their proliferative capacity (5).
Furthermore, we had demonstrated an ability of PMA to bypass the defect
in anergic cells that otherwise resulted in an inability to activate
MAPK. Therefore, it was of interest to determine whether the repressor
molecule acting in anergic murine/murine heterokaryons had any capacity
to interfere with IL-2 protein accumulation in response to treatment
with Iono/PMA. If true, this would indicate that the repressor molecule
acts downstream of the MAPK or in a separate signaling pathway parallel
to the MAPK cascade. IL-2 production in anergic murine/murine
heterokaryons was, in fact, observed to be defective following
incubation in the presence of Iono and PMA (Fig. 5C).
Anergic murine/murine heterokaryons activated with Iono/PMA again
demonstrated a 48 ± 5% reduction in IL-2 production relative to
that by control heterokaryons.
The finding that anergic heterokaryons remain defective for IL-2
synthesis even after stimulation with the combination of a calcium
ionophore and protein kinase C-activating phorbol ester suggests that
clonal anergy does not act solely to interfere with signals upstream of
ERK and JNK. Consistent with this, we observed that intracellular IL-2
accumulation by anergic DO11.10 T cells was only partially restored
during stimulation with Iono/PMA (38% of the normal control T cell
response vs a response that was 7% of the control following
stimulation with CD3/CD28 mAbs; Fig. 6).
Taken together, these results now suggest a model in which clonal
anergy induction both interferes with the upstream activation of
p21ras and the MAPK and represses the activation
of the IL-2 gene at a point either downstream of or parallel
to the MAPK. Thus, a biochemical examination of signal transduction
events in anergic heterokaryons will be necessary to determine whether
this negative regulator is also acting to block signal transduction
upstream of p21ras or whether complementation of
a proximal defect did, in fact, occur in the setting of a coexisting
molecular repressor.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Daniel L. Mueller, Center for Immunology, University of Minnesota Medical School, Box 334 FUMC, 312 Church St. S.E., Room 6-120 BSBE, Minneapolis, MN 55455. E-mail address:
3 Abbreviations used in this paper: PEG, polyethylene glycol; Iono, ionomycin; HPRT, hypoxanthine phosphoribosyltransferase; CFSE, carboxyfluorescein diacetate/succinimidyl ester; MAPK, mitogen-activated protein kinase.
Received for publication August 10, 1998. Accepted for publication October 26, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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