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IMMUNOBIOLOGY
From the Department of Research, University Hospital
Basel, and Institute of Anatomy, University of Basel, Basel,
Switzerland.
The flt3 ligand (FL) is a growth and differentiation factor for
primitive hematopoietic precursors, dendritic cells, and natural killer
cells. Human T lymphocytes express FL constitutively, but the cytokine
is retained intracellularly within the Golgi complex. FL is mobilized
from the cytoplasmic stores and its serum levels are massively
increased during the period of bone marrow aplasia after stem cell
transplantation (SCT). Signals that trigger the release of FL by T
cells remain unknown. This study shows that interleukin (IL)-2, IL-4,
IL-7, and IL-15, acting through a common receptor T lymphocytes are central to the immune responses
after allogeneic stem cell transplantation (SCT). Attack by donor T
cells against recipient alloantigens may cause graft-versus-host
disease.1 On the other hand, T cells can improve the
transplantation outcome by preventing graft rejection and reducing the
incidence of leukemic relapse.2,3 The mechanisms by which
T cells promote engraftment and confer antitumor reactivity after SCT
are only partly understood.
The interaction of interleukin (IL)-2 with the IL-2 receptor (IL-2R)
plays an important role in regulating the magnitude and duration of
T-cell-dependent immune responses.4 High-affinity IL-2R
comprises subunits The flt3 ligand (FL) is a hematopoietic cytokine with a broad range of
activities at early stages of hematopoiesis.14,15 In
synergy with other hematopoietic growth factors, FL stimulates the
self-renewal and proliferation of multipotent and lineage-committed progenitors.16 Remarkably, FL is important for the
development of hematopoietic precursors into functionally mature DCs
and NK cells.17,18 DC and NK cell numbers in the lymphoid
organs and circulation are severely reduced by targeted disruption of
the FL gene,19 whereas they are increased after FL
administration.20,21 Treatment of tumor-bearing mice with
FL leads to potent antitumor responses mediated by DCs and NK
cells.13,22 FL is expressed by T cells and bone marrow
stroma cells as membrane-bound FL (mFL) that undergoes a subsequent
processing to generate soluble FL (sFL).23 We have
recently demonstrated that production of FL by T cells is dependent on
the status of the stem cell compartment. During normal hematopoiesis,
FL is expressed constitutively but most of it is retained
intracellularly within the Golgi apparatus. During bone marrow aplasia
preceding the engraftment of transplanted stem cells, FL is released
from cytoplasmic stores and its serum levels are highly elevated,
suggesting a role for FL in restoring normal
hematopoiesis.24,25 Signals that can trigger the release of FL from T cells remain unknown and are the subject of this study.
Here we show that FL expression by human peripheral blood T lymphocytes
is significantly up-regulated by IL-2, IL-4, IL-7, and IL-15. This
process is mediated by the common Reagents, cytokines, and IS drugs
Purification and activation of T cells
Patients The study included 37 patients with different hematologic malignancies (21 with acute myeloid leukemia, 4 with chronic myeloid leukemia, 5 with myelodysplastic syndrome, 2 with multiple myeloma, 2 with acute lymphocytic leukemia, 2 with non-Hodgkin lymphoma, and 1 with Burkitt lymphoma). There were 22 males and 15 females; the median age was 54 years (range, 4-70). Nineteen patients had been treated with chemotherapy, 11 patients had undergone allogeneic SCT, and 7 patients had had autologous SCT. All recipients of allogeneic grafts were being treated with CsA (120-300 mg/d). Blood samples were collected on days 10 to 19 after the beginning of treatment; the leukocyte count at that time was 0.28 ± 0.02 × 109/L. Peripheral blood was obtained with informed consent in compliance with the guidelines of the Ethical Committee of the University Hospitals of Basel (Basel, Switzerland).Enzyme-linked immunosorbent assay (ELISA) To obtain serum, patients' native peripheral blood was centrifuged for 10 minutes at 3000 rpm. T-cell culture supernatants were collected after 72 hours of cell activation. Samples were aliquoted and stored at 70°C until use. Soluble FL was measured by
ELISA (IMMUNEX, Seattle, WA)27; the reaction was linear in the range 6.25 to 200 pg/mL.
Flow cytometry (FACS) For analysis of mFL in purified resting or activated T lymphocytes, cells were washed in a FACS buffer containing phosphate-buffered saline (PBS), 0.5% bovine serum albumin, and 0.02% NaN3 and incubated for 20 minutes on ice with anti-FL mAb M5 (rat IgG2a, IMMUNEX) or control rat IgG2a (PharMingen) followed by fluorescein isothiocyanate (FITC)-conjugated goat antirat IgG (Jackson Immunoresearch Lab, West Grove, PA). For analysis of mFL in patients' peripheral blood, double-color staining of mononuclear cells with anti-FL mAb M5 (as above) and phycoerythrin-conjugated anti-CD3 mAb (Becton Dickinson, San Jose, CA) was performed. Acquisition was done using a FACScan (Becton Dickinson). Dead cells were stained with propidium iodide and excluded from the analysis; 10 000 to 15 000 events were acquired. Analysis was performed using CellQuest software (Becton Dickinson). Expression of mFL is given as median fluorescence intensity (MFI) ratio of signals produced by specific and isotype-matched control mAbs.Confocal microscopy Following activation, cultured T cells were separated on Histopaque 1077 density gradient (Sigma) to remove dead cells. Immunostaining was performed as described elsewhere.24 Briefly, cells were allowed to settle onto Poly-L-Lysin-coated coverslips (Sigma), fixed with 4% paraformaldehyde, and permeabilized with 0.1% saponin. Unspecific staining was blocked with 0.5% sodium borohydride followed by incubation with a 5% mixture of goat and donkey sera. Slides were incubated for 1 hour at room temperature with mAb M5 or control rat IgG2a and antibodies against human giantin (mouse IgG1; kind gift of H.-P. Hauri, Biozentrum, Basel, Switzerland), TGN46 (rabbit polyclonal antibody; kind gift of S. Ponnambalam, University of Dundee, UK), or calnexin (rabbit polyclonal antibody; kind gift of A. Helenius, University of Zürich, Zürich, Switzerland). Cells were stained for 1 hour in the dark with the following secondary antibodies showing no species cross-reactivity: donkey antirat IgG-cyanin(Cy)2, goat antimouse IgG-TexasRed, goat antirabbit IgG-TexasRed (all from Jackson Immunoresearch Lab), goat antirabbit IgG-Cy3 or goat antimouse IgG-Cy5 (both from Amersham, Pittsburgh, PA). Slides were mounted with Mowiol containing 20 mg/mL 1,4-diazabicyclooctane (DABCO; Sigma). Confocal microscopy was performed with TCS4D (Leica, Glattbrugg, Switzerland) or with LSM 510 (Carl Zeiss AG, Jena, Germany) operating in the sequential acquisition mode to exclude cross-talk between channels and using 488, 568, and 647 excitation lines. Images were analyzed for colocalization using the IMARIS software package (Bitplane AG, Zürich, Switzerland).RNase protection assay The pBluescript vector containing human FL clone 9 cDNA28 was obtained from S. Lyman (IMMUNEX). A region of complementary DNA (cDNA) coding for multiple alternatively spliced transcripts was removed after digestion with BamHI (in T3 polylinker) and NcoI (position 58514 in the FL cDNA insert). Plasmid was religated with T4DNA ligase, linearized with XhoI and an [ -32P]UTP-labeled antisense RNA probe was synthesized
using the MAXIscript kit (Ambion, Austin, TX). Total RNA (10 µg) was
hybridized with FL antisense probe and an RNase A/T1
protection assay was performed using the HybSpeed RPA kit (Ambion)
according to the manufacturer's instructions. Two micrograms RNA was
hybridized with the  -actin antisense probe (template provided by
Ambion). Protected RNA fragments were resolved on a 5% denaturing
polyacrylamide gel and autoradiographed for 7 days (FL) or 3 hours
(-actin) at 70°C using Kodak x-ray film (Eastman Kodak,
Rochester, NY) and intensifying screens. Protected RNA fragments were
quantified using a PhosphorImager and ImageQuant software (both from
Molecular Dynamics, Sunnyvale, CA). Size markers were obtained by
digestion of pBR322 with NarI and DdeI (for FL) or with HinfI (for
-actin), followed by [-32P]ATP-labeling with Klenow
DNA polymerase (Promega, Madison, WI).
Statistics The nonparametric Mann-Whitney U test was applied to analyze differences in FL expression in vitro and in patient groups. Differences were considered statistically significant at P < .05.
Cytokines using the common or IFN- did not increase mFL above the level seen
without cytokine addition (Figure 1D) and had no effect on responses
mediated by IL-2 or IL-7 (not shown). The specificity of cytokine
effects on mFL expression was confirmed by measuring sFL in culture
supernatants. No increase above the spontaneously released amount was
observed in the presence of TNF-, IFN-, IL-6, or GM-CSF, whereas
IL-2 and IL-7 increased sFL levels 3- to 5-fold above background
(Figure 1E). A similar increase was also obtained in response to IL-4
and IL-15 (not shown). These results indicate that IL-2, IL-4, IL-7,
and IL-15, that is, cytokines using a common c receptor subunit, can
induce cell surface expression and release of FL by T lymphocytes. To confirm the involvement of c signaling in regulation of FL
expression, we used anti-c mAb CP.B8, which blocks c association
with other receptor subunits,26 and tyrphostin AG490,
which prevents activation of Jak3, a tyrosine kinase that associates
with c on ligand binding.29 As shown in Figure
2, the addition of either CP.B8 or
tyrphostin AG490 resulted in a dose-dependent inhibition of the IL-7
effect on mFL and sFL expression.
To gain some insight into the mechanism of
Taken together, these results indicate that triggering of the T-cell activation by CD3 and CD28 ligation enhances proteolytic cleavage of mFL To examine the possible involvement of TCR signaling in FL expression, we incubated T cells for 72 hours with the plate-immobilized anti-CD3 mAb in the presence of mAb against the costimulatory receptor, CD28. RNase protection analysis revealed that activation of T cells by CD3 and CD28 ligation lowers the expression of FL mRNA by about 10-fold (Figure 3). This finding is consistent with the observation that treatment with concavalin A strongly decreases FL mRNA expression in a murine T-cell line.15 Next, we examined FL protein expression in CD3/CD28-stimulated cells (Table 1). Levels of mFL were below those seen for control cells cultured without stimulation (MFI, 1.6 ± 0.1 versus 2.5 ± 0.1), whereas sFL levels were increased from 24.2 ± 2.2 to 62.8 ± 2.5 pg/mL. A similar increase in sFL, but not mFL, was found when CD3/CD28 stimulation was performed in the presence of IL-7. This is in sharp contrast to a significant increase in both FL forms in response to IL-7 alone. We have verified that the effect brought about by receptor stimulation was not due to changes in the proportion of FL-specific transcripts: Using PCR primers that allow a distinction to be made between alternative transcripts encoding mFL and sFL,15,28 we found no increase in sFL transcripts in cells activated by CD3 and CD28 ligation compared to IL-7 treatment (results not shown).
We investigated the possibility that activation of T cells
through CD3 and CD28 receptors enhances the proteolytic cleavage of mFL
at the cell surface and thus augments the release of sFL into culture
supernatants. We used PMA, an inducer of proteinases involved in
cleavage of membrane-associated cytokines.30 PMA treatment
of T cells for 3 hours perfectly mimicked the effect of CD3/CD28
stimulation (Table 1 and Figure 4).
Moreover, like CD3 and CD28 ligation, PMA decreased the
IL-7-induced mFL levels from MFI of 7.7 ± 0.4 to 1.6 ± 0.1,
whereas sFL increased to as much as 146.2 ± 11 pg/mL. The effect of
PMA was prevented by addition of the metalloproteinase inhibitor BB3103
(Figure 4), suggesting that metalloproteinase(s) play a role in
proteolytic processing of mFL.
These results indicate that TCR-dependent T-cell activation has a dual effect on FL expression, by decreasing the level of FL mRNA and enhancing the proteolytic cleavage of mFL. CsA inhibits the
Intracellular trafficking plays an important role in the regulation of
FL expression by T cells.24 We examined the intracellular distribution of FL in T cells treated with IL-7 and CsA. Confocal microscopy was used to visualize colocalization of FL with organellar markers (Figure 6). In agreement with our
previous findings, untreated "ex vivo" T cells contained a cluster
of preformed FL visible as a strong signal within and close to the
Golgi apparatus (Figure 6A,B). FL immunofluorescence signals partially
overlapped with those of giantin, a Golgi marker,35 and to
a larger degree with TGN46, a marker of the trans-Golgi
network.36 In cells treated with IL-7, the bulk of FL
still overlapped with Golgi markers, but FL signals were also scattered
through the cell cytoplasm and were visible at the outer rim of the
cell (Figure 6C,D), indicative of the release of FL from intracellular
stores in response to cytokine stimulation. Importantly, these
scattered signals were absent in cells treated with CsA (Figure 6E,F),
suggesting that the drug prevented the release of FL from the Golgi
apparatus. Furthermore, the overlap of FL with the resident Golgi
proteins, depicted as a yellow signal, was more pronounced with giantin (Figure 6E) than with TGN46 (Figure 6F). Indeed, colocalization analysis revealed that in cells treated with CsA, only 19% of FL
staining colocalized with TGN46, whereas in IL-7-treated cells, the
colocalization of FL with TGN46 was 64%. These results demonstrate that IL-7 induces trafficking of FL to the cell surface, and that this
process is inhibited by CsA, most prominently at the level of
transition from the main body of the Golgi apparatus to the trans-Golgi
compartment. Additional evidence that exocytosis from the Golgi complex
plays an important role in determining FL expression in T cells was
provided by the use of BfA, a reagent that blocks protein secretion by
disrupting the Golgi and redistributing Golgi proteins to the
endoplasmic reticulum (ER).37 BfA fully abrogated mFL
expression in response to IL-2 (Figure 6G). Confocal microscopy analysis of cells treated with IL-2 and BfA confirmed that both giantin
and FL signals were completely disrupted and colocalized to a large
extent with the ER marker, calnexin (Figure 6H).
CsA decreases FL levels in patients undergoing allogeneic SCT We have previously shown that FL expression increases in patients undergoing myeloablative treatment, irrespective of the underlying malignancy.24,25 To assess the influence of CsA on FL expression in vivo, we compared levels of mFL in T cells and sFL in serum in 3 groups of patients differing with respect to CsA use during therapy. Patients in group 1 were treated with chemotherapy alone, patients in group 2 underwent autologous SCT, and patients in group 3 underwent allogeneic SCT and received CsA for prevention of graft-versus-host disease. At the time of FL monitoring, all patients were severely pancytopenic, with white blood cell count of 0.28 ± 0.02 × 109/L compared to 3.5 to 10.0 × 109/L in healthy subjects. In all 3 patient groups, levels of mFL and sFL were significantly elevated above normal (Figure 7). However, average level of mFL was about 2-fold lower in patients treated with CsA than in control groups not receiving CsA (mean ± SEM; 3.7 ± 0.7 compared to 7.1 ± 0.6 and 5.3 ± 0.8; P < .005). Similarly, sFL levels were decreased in patients treated with CsA (mean ± SEM; 919 ± 252 compared to 1571 ± 140 and 1817 ± 205 pg/mL; P < .01). Despite a considerable overlap of values in the 3 groups, FL levels in 4 of 11 patients receiving CsA were nearly as low as normal. These results are in accordance with an inhibitory effect of CsA on FL expression by T cells in vitro and show that CsA treatment partly counteracts the up-regulation of FL in response to stem cell deficiency in vivo.
Owing to a capacity to expand both the primitive hematopoietic
progenitors and DCs and NK cells,16,20,21 FL may play an important role during hematopoietic and immune recovery after SCT, a
hypothesis supported by a rapid and massive increase in circulating FL
in the immediate posttransplantation period.24 T cells are
the major source of FL during aplasia preceding the engraftment of stem
cells, but signals that up-regulate FL levels remain unknown. We used T
cells purified from normal human peripheral blood to elucidate the
T-cell-specific ligands and molecular mechanisms that control
expression of FL. The results demonstrate that signaling mediated by
the cytokine receptor Interleukin-2, IL-4, IL-7, and IL-15, the cytokines sharing the common
Our previous studies demonstrated that in patients undergoing SCT,
up-regulation of FL in response to stem cell deficiency is due to rapid
mobilization of preformed FL from cytoplasmic stores in T cells,
followed by a delayed transcriptional activation of the FL
gene.24 Given the similarities in FL regulation occurring in vivo and in response to The unexpected finding of this study is that transcription of the FL gene is down-regulated by TCR signaling brought about by engagement of CD3 and CD28 receptors. This effect distinguishes the regulation of FL from that of other known cytokine-encoding genes, whose transcription is initiated in response to signals transduced by the TCR complex together with signals provided by costimulatory molecules.50 The amount of IL-2 released by T cells activated through CD3 and CD28 receptors is about 3 ng/1 × 106 cells51 and, thus, sufficient to up-regulate FL. However, a decrease in FL transcript levels in response to TCR signaling indicates that activated cells can avoid the possibility that enhanced expression of IL-2 is always coupled to up-regulation of FL. Our data also show that CD3 and CD28 receptor stimulation affects expression of FL at the posttranslational level, by increasing the proteolysis of mFL and release of sFL to culture supernatants. Enhanced proteolysis of FL may be due to up-regulation of matrix metalloproteinases on cell activation.52 Accordingly, this effect was mimicked by PMA, which activates enzymes that shed membrane-bound cytokines. Putative metalloproteinase(s) responsible for FL cleavage have not been identified, yet our results argue for a link between TCR activation and FL protein processing. Finally, we found that CsA inhibits Several aspects of our findings may be relevant for understanding the
mechanisms of hematopoietic and immune reconstitution after SCT. A link
between In conclusion, our results demonstrate that FL belongs to the
We thank S. D. Lyman for FL-related reagents; H.-P. Hauri, A. Helenius, S. Ponnambalam, and D. Baker for antibodies; W. Schuler for immunosuppressive drugs; A. Gratwohl, J. Passweg, and C. Pino for providing patient blood samples; and G. De Libero, C. Kalberer, and A. Luther for helpful comments on the manuscript.
Submitted July 20, 2000; accepted October 24, 2000.
Supported by grants from the Swiss National Science Foundation (32-055694.98), Swiss Cancer League (KFS 951-09-1999), and Roche Research Foundation (to O.P.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Aleksandra Wodnar-Filipowicz, Research Department, University Hospital Basel, Hebelstrasse 20, CH-4031 Basel, Switzerland; e-mail: aleksandra.wodnar-filipowicz{at}unibas.ch.
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| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
-chain signaling
and inhibited by cyclosporin A
Top
Abstract
Introduction
mAb TR66 (kind gift of G. Spagnoli, University of
Basel, Switzerland) at 20 µg/mL for 16 hours. Activation with
plate-immobilized TR66 mAb in the presence of mouse antihuman CD28 mAb
(PharMingen, San Diego, CA) at 2 µg/mL was for 72 hours.
a possible role of high endogenous IL-2 in their suppression.
Acta Haematol.
1999;101:165-172
cells with preserved ability to engraft SCID-hu bone.
Blood.
1998;91:1206-1215