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Is Reversed by IL-151
*
Academic Transfusion Medicine Unit, and
Centre for Rheumatic Disease, Department of Medicine, University of Glasgow, Royal Infirmary, Glasgow, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, a cytokine
widely implicated in suppression of inflammatory responses and secreted
by many different tumor cell types. There have been conflicting reports
regarding inhibition of IL-2-induced STAT3 and STAT5 phosphorylation by
TGF and subsequent suppression of immune responses. Using
TGF-producing multiple myeloma tumor cells we demonstrate that
tumor-derived TGF can block IL-2-induced proliferation and STAT3 and
STAT5 phosphorylation in T cells. High affinity IL-2R expression was
required for the suppression of IL-2 responses as a novel
CD25- T cell line proliferated and phosphorylated
STAT3 when cultured with tumor cells or rTGF1. Activating T cells
with IL-15, which does not use the high affinity IL-2R, completely
restored the ability of T cells to phosphorylate STAT3 and STAT5 when
cultured with tumor cells. IL-15-treated T cells proliferated normally
when cocultured with tumor cells or rTGF1, whereas IL-2 responses
were consistently inhibited. Preincubation with IL-15 also restored the
ability of T cells to respond to IL-2 by phosphorylating STAT3 and
STAT5, and proliferating normally in the presence of tumor cells. IL-2
pretreatment did not restore T cell function. IL-15 also restored T
cell responses by T cells from multiple myeloma patients, and against
freshly isolated bone marrow tumor samples. Thus, activation of T cells
by IL-15 renders T cells resistant to suppression by TGF1-producing
tumor cells and rTGF1. This finding may be exploited in the design
of new immunotherapy approaches that will rely on T cells avoiding
tumor-induced suppression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, which is produced by a
diverse range of tumor cell types, such as breast carcinoma, renal cell
carcinoma, Hodgkin’s lymphoma, and multiple myeloma
(MM),3 inter alia
(1, 2, 3, 4, 5). TGF has a wide spectrum of
anti-inflammatory properties and suppresses the production and
actions of mediators of cell-mediated immunity such as IL-2, IL-12, and
IFN-
(6, 7, 8). TGF also suppresses killing activity of
cytotoxic T cells and NK cells, and is the principal effector produced
by Th3 or Tr1 suppressor T cells (9, 10) (in conjunction
with IL-10 in the latter subset).
TGF blocks T cell expansion in response to Ag, as it is a potent
inhibitor of the autocrine IL-2 pathway. TGF induces a
G1 cell cycle arrest in IL-2-responsive T cells
and stops IL-2 production, and T cells essentially become anergic
(6). The exact effects that TGF has on IL-2 signaling
remain controversial. High affinity IL-2 receptors signal through Janus
kinase (Jak)1/STAT5 and Jak3/STAT3 leading to cell proliferation and
production of IL-2 (6, 11). Murine TGF1 has originally
been reported to inhibit both Jak1 and Jak3 and subsequent STAT3 and
STAT5 phosphorylation (7). This was contradicted by a
separate study that found only Jak1/STAT5 inhibition (6)
(although this used TGF2), and a recent study using human cells
found no modulation of STAT5a function by TGF1 (8).
These differences may represent different requirements of murine and
human T cells in proliferation and cytokine production.
One aim of studies into IL-2 signaling defects induced by TGF is to
overcome TGF-mediated immune suppression by tumors. In previous
investigations into MM plasma cell tumors, we have shown that TGF is
wholly responsible for suppression of IL-2-induced T cell proliferation
by the tumor cells (1). However, T cell blasts, which
lacked the IL-2R
(CD25) chain and relied on exogenous rather than
autocrine IL-2, were found to be resistant to tumor cells and rTGF
(1). This suggests that TGF suppression of IL-2
responses can be influenced by whether the T cells use intermediate
affinity (CD122/CD132) or high affinity
(CD25/CD122/CD132) IL-2 receptors. Therefore, activation of T cells by
cytokines that do not require the apparently TGF-sensitive high
affinity IL-2 receptor could potentially overcome TGF-mediated
immune suppression.
IL-15 is a T cell-stimulatory cytokine, primarily monocyte derived,
which has many properties in common with IL-2 (12, 13).
IL-15 enhances T cell proliferation and also shares the and
subunits of the IL-2 receptor (although IL-15 uses a unique subunit
(IL-15R) to form high affinity receptors) (14). IL-15
also induces the phosphorylation of STAT3 and STAT5 (11, 15). However, IL-15 has a number of properties that distinguish
it from IL-2. The induction of IL-2 and T cell activation by IL-15 is
initially independent of the autocrine loop through IL-2/CD25
(14), although activated cells subsequently enter this
pathway. IL-15 can enhance T cell activation where IL-2 pathways are
suppressed, e.g., in HIV (16). Also, IL-15 can protect T
cells from a variety of apoptosis-inducing agents where IL-2 is
ineffective (17).
The ability of IL-15 to maintain T cell activation and IL-2 responses
is of great potential in activating T cells where IL-2 responses are
suppressed. Recombinant or tumor-derived TGF suppresses the ability
of T cells to make and respond to IL-2. Therefore, we examined the
ability of IL-15 to maintain T cell activation in the face of
recombinant or MM tumor cell-derived TGF-induced suppression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anti-human CD3, CD4, CD8, CD25, CD38, and CD69 mAbs conjugated
with FITC or PE were obtained from PharMingen/BD Biosciences (Abingdon,
U.K.). Anti-STAT3, phospho-STAT3, and STAT5 (recognizes STAT5
a+b) mAbs were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). Anti-phospho-STAT5 was purchased from New England Biolabs
(Beverly, MA). Anti-BB4-PE (CD138) was purchased from Serotec (Oxford,
U.K.). "Pansorbin" Staphylococcus aureus cells (SAC)
were purchased from Calbiochem (La Jolla, CA). Human (h)rIFN-,
rIL-12, and rTGF1 were purchased from R&D Systems (Minneapolis, MN).
Human rIL-2 (sp. act. 4 U/ng), staphylococcal enterotoxin B
(SEB), and latency-associated peptide (LAP) were purchased from
Sigma-Aldrich (St. Louis, MO). Human rIL-15 (sp. act. 177 U/ng) was
obtained from Immunex (Seattle, WA).
PBMC
Venous blood was collected from healthy volunteers or MM patients in plateau phase of disease following informed consent, using sodium heparin as anticoagulant. PBMC were separated over Ficoll/Hypaque (Nycomed, Oslo, Norway) and washed four times in PBS.
Cell lines
The MM cell lines U266 and JIM1 were used in these experiments. Cell lines were maintained in complete tissue culture medium: RPMI 1640 medium, 10% FCS, 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin.
The hIL-2-dependent T cell line BDB2 (generated in this laboratory, originally named IDB; Ref. 1) was maintained in complete tissue culture medium supplemented with 20 U hrIL-2.
MM samples
Samples from myeloma patients were obtained following informed consent. Heparinized bone marrow (BM) aspirate cells from MM patients were cultured overnight in RPMI 1640 medium supplemented with 10% autologous BM plasma, glutamine, and antibiotics. Dead cells were removed by centrifugation over Ficoll-Hypaque, and the relative percentage of CD38++/BB4+ tumor cells was determined by FACS analysis.
Mitomycin C treatment
MM cell lines or BM cells to be used in MLCs were treated with mitomycin C (Sigma-Aldrich). Cells (2 x 107/ml) were treated with 50 µg/ml mitomycin C in complete medium for 30 min at 37°C, followed by two washes in RPMI 1640 medium.
T cell activation assays
PBMC (4 x 106/ml) were incubated with mitomycin C-treated tumor cells to give tumor-PBM ratios of 1:5. Samples were stimulated with 2.5 µg/ml Con A ± 10 ng/ml hrIL-2 or 10 ng/ml IL-15. Samples for flow cytometry were cultured in 24-well (2 ml/well) plates. Samples for proliferation assays were cultured in quadruplicate in 96-well microtiter plates, and proliferation was assessed by the addition of 1 µCi [3H]dThd in the last 6 h of incubation. Incorporated radiolabel was assessed in a Packard (Meriden, CT) Matrix 96 counter.
Soluble (s)IL-15R assays
To assess the impact of sIL-15R (18) on T cell
activation, 5 ml of PBMC in complete medium was adhered to plastic for
2 h at 107 cells/ml in a six-well plate. The
nonadherent cells were then removed and stored at 37°C in the
complete culture medium. Adherent cells were either cultured in medium
alone or stimulated with 100 U/ml IFN- and 5 µl/ml of 10% SAC
solution for 18 h, followed by five washes in PBS. The nonadherent
cells were then returned to their originating wells and
stimulated with SEB 5 µg/ml ± 140 ng/ml sIL-15R ± U266
cells (1:5 ratio) for up to 5 days. Activation of T cells was assessed
as described below. Control sIL-15R was generated by incubating
sIL-15R at a ratio of 400:1 with hrIL-15 for 30 min at 4°C. These
concentrations have been found to block IL-15 binding sites on the
receptor without leaving sufficient residual IL-15 in the preparation
to mediate any IL-15-induced proliferation (results not shown).
T cell restimulation assay
The ability of normal T cells to respond to IL-2 or IL-15 was assessed as previously described (1, 6). Briefly, PBMC (4 x 106/ml) were stimulated with 5 µg Con A for 5 days ± 10 ng/ml IL-2 ± 10 ng/ml IL-15. The cells were then washed twice in complete medium followed by 24-h culture in complete medium alone containing 1% FCS. The cells were then purified over Ficoll-Hypaque, washed twice in complete medium (resulting cells were >98% CD3+, results not shown), and restimulated in complete medium containing various stimuli as detailed in Results. Proliferation was assessed by [3H]dThd counting.
Flow cytometry
Cells (5 x 105/sample) were stained by direct immunofluorescence for two-color flow cytometry using isotype-matched controls. Data were analyzed using CellQuest software (BD Biosciences, Mountain View, CA).
Western blotting
Western blotting was conducted using standard techniques. BDB2 cells or activated/rested T cells were restimulated for 30 min with medium alone, 10 ng/ml IL-2, or IL-15 ± U266 1:5. Cells were also incubated with tumor cells, IL-2, or IL-15 and LAP 80 ng/ml. Identical samples were also assessed after 15-, 30-, 60-, and 140-min stimulation. P-STAT3 and P-STAT5 levels were assessed by Western blotting overnight with 2 µg/ml appropriate Ab. Bands were detected with anti-mouse Ig-HRP or anti-rabbit Ig-HRP, respectively. Gels were visualized by chemiluminescence (ECL; Pharmacia Biotech, Uppsala, Sweden). Blots were stripped in 62.5 mM Tris-HCl pH 6.7 containing 2% SDS/100 mM 2-ME at 50°C for 20 min, and reprobed for STAT3 and STAT5 levels. Bands were scanned at 900 dots per inch and quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). P-STAT data were expressed in arbitrary units, normalized against total STAT protein detected.
Statistical analysis
Where appropriate, statistical analysis was performed using a paired t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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from MM cells inhibits IL-2-induced STAT3 and STAT5
phosphorylation
It has been previously shown that MM cells inhibited IL-2-induced
T cell proliferation, and that the inhibitory agent was TGF, as
responses could be restored by addition of LAP (1). Here
we examined the signaling defects associated with MM cell-induced
suppression of IL-2 responses. IL-2 and IL-15, when used at 10
ng/ml, had been previously found to induce comparable proliferative
responses and levels of STAT3 and STAT5 activation in T cells in our
hands, and were used as the standard dose throughout. MM cells were
used at a standard dose of one MM cell per five T cells as this had
previously been demonstrated to provide an approximate
ED75 for inhibition of T cell proliferation to
IL-2 (1). Other studies indicated that STAT3 and STAT5
phosphorylation first appears after 15 min of IL-2 treatment and is
stable for at least 60 min, thus STAT activation was examined at 30 min
in these studies. Activated T cells phosphorylated STAT3 and STAT5 when
restimulated with 10 ng/ml IL-2, but this phosphorylation was
completely blocked by the addition of U266 cells at the 1:5 ratio.
However, T cells were able to phosphorylate STAT3 and STAT5 when
TGF-LAP was added to the cultures at
80 ng/ml (Figs. 1
a and
2, a and b). Thus,
TGF was responsible for the MM cell-induced failure of T cells to
phosphorylate STAT3 and STAT5. Examining STAT3 over a 4-h time period
confirmed that IL-2 induced maximal phosphorylation at 30 min, which
declined by 4 h. U266 cells suppressed all phosphorylation during
this time period, but LAP was effective in maintaining phosphorylation
at normal levels. Thus, the TGF-blocking effects of LAP were
effective throughout the period of IL-2-induced STAT3 phosphorylation.
The suppressive effect of the myeloma cells was proportional to the
numbers of cells added to the T cells; a dose of 1 MM per 20 T cells
still inhibited STAT3 and STAT5 phosphorylation, but not completely
(Figs. 1b and 2b).
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Normal T cells stimulated with IL-15 phosphorylated STAT3 and
STAT5. In contrast to IL-2-induced signaling, addition of U266 tumor
cells did not lead to any reduction in STAT3 or STAT5 phosphorylation
by IL-15-stimulated T cells (Figs. 1
a and 2a).
IL-15 has been demonstrated to enhance IL-2 responses, and this was
examined by stimulating normal T cells with 10 ng/ml IL-2 or IL-15
followed by restimulation with IL-2 ± U266 tumor cells. No
differences were detected between T cell cultures treated with IL-2 and
IL-15 in the numbers of CD3, CD4, and CD8 cells or their levels of
expression of CD25 (results not shown). T cells pretreated with IL-2
readily phosphorylated STAT3 and STAT5 in response to IL-2. However,
this was greatly reduced by the addition of U266 cells (Figs. 1d and 2c). Pretreatment of T cells with IL-15
overcame this block on STAT3 and STAT5 phosphorylation, with
IL-15-pretreated cells successfully phosphorylating STAT3 and STAT5 in
response to IL-2 (Figs. 1e and 2d).
BDB2 T cell proliferation is associated with STAT3 but not STAT5 phosphorylation
The BDB2 T cell line is IL-2 dependent, and does not make IL-2 or
express CD25, therefore residing outside the autocrine IL-2 pathway
(1). This cell line is not inhibited in its IL-2 responses
by MM cells (1) or rhTGF1 (data not shown). When the
BDB2 cell line was restimulated with IL-2, both STAT3 and STAT5 were
phosphorylated (Fig. 3). In the BDB2
cells, addition of U266 cells completely abrogated STAT5
phosphorylation (Fig. 3b) but STAT3 phosphorylation was not
inhibited (Fig. 3a). Thus, STAT3 phosphorylation was
required for BDB2 T cells to proliferate when cocultured with the tumor
cells, but STAT5 was not.
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The effects that IL-15 had on T cell activation and proliferation
when cocultured with tumor cells were assessed. Fresh peripheral blood
T cells cocultured with MM cell lines are deficient in the ability to
up-regulate IL-2R (CD25) in response to mitogens (1).
Here, normal peripheral blood T cells were cocultured with U266 (1:5)
and activated with Con A ± 10 ng IL-2 or IL-15 for 72 h. No
differences in the levels of CD25 expressed by positive control
cultures incubated with Con A and IL-2 or IL-15 were detected (data not
shown). Although the tumor cells significantly reduced CD25 expression
by IL-2-stimulated cells, IL-15 restored this expression to levels
identical to positive controls (Fig. 4a). Also, when normal T cells
were incubated with Con A ± IL-2 or IL-15 and fresh BM tumor
cells or U266 cells, IL-15 maintained proliferation, whereas IL-2
responses were inhibited (Fig. 4, b and c).
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a). Addition of the MM cell
lines significantly decreased proliferation to IL-2, whereas no
significant decrease in proliferation was detected in IL-15-stimulated
cells (Fig. 5a). These experiments were repeated using
rTGF1, and essentially identical results were obtained. TGF1
significantly suppressed T cell proliferation to IL-2 but had no
suppressive effect on IL-15; indeed, there was some evidence of synergy
between TGF1 and IL-15 in inducing T cell proliferation (Fig. 5b).
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IL-15 is produced by activated monocytes (12). To
test whether native monocyte-derived IL-15 could protect T cells from
suppression during a primary proliferative response, resting and
SAC/IFN--activated monocytes were cocultured with SEB-activated T
cells ± U266 cells. Upon primary stimulation, T cells usually
progress through a CD69/CD25 double-positive phase, ultimately becoming
singly CD25 positive. T cells that have been inhibited by coculture
with MM cells fail to up-regulate CD25 and remain arrested at the
CD69-positive stage. SEB-activated cells containing normal monocytes
had become largely CD25 positive by day 5 with few CD69-positive cells,
but addition of U266 cells led to the characteristic arrest of the T
cells at the CD69-positive stage (Fig. 6, a and b). Activated monocytes reversed this
inhibition by U266 cells, and the T cells moved to the
CD69-/CD25+ stage and were
essentially indistinguishable from T cells activated without the
addition of U266 (Fig. 6c). The protective effect of the
monocytes appeared to be mediated by IL-15, as addition of sIL-15R
to the cultures led to the T cells displaying the characteristic
arrested CD69-positive phenotype (Fig. 6d). Soluble
IL-15R preabsorbed with IL-15 was not effective at reversing the
protective effect of inflammatory monocytes, and T cells displayed
normal activation markers (results not shown). Thus, natural IL-15 from
monocytes was effective at maintaining T cell activation in the
presence of myeloma cells.
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As pretreatment with IL-15 had been shown to restore the ability
of T cells to phosphorylate STAT3 and STAT5 in response to IL-2, the
effects that this had on proliferation were examined. As both IL-2 and
IL-15 mediate T cell proliferation by up-regulating CD25 and IL-2
production, the ability of IL-15 to maintain T cell proliferation may
be due to IL-15-stimulated T cells having increased sensitivity to
IL-2. To test this hypothesis, PBMC were activated with the addition of
10 ng IL-2 or 10 ng IL-15 during the initial activation period, then
restimulated with reducing concentrations of IL-2 ± U266 cells.
IL-2-pretreated T cells responded in a dose-dependent manner to IL-2,
with a plateau at 20–10 ng, whereas IL-15-pretreated T cells
maintained virtually identical proliferation rates over the range
tested (Fig. 7, a and
b). This increased IL-2 sensitivity was significant in
protecting against MM suppression, with IL-15-pretreated cells able to
respond well to IL-2, whereas IL-2-pretreated cells showed poor
proliferation (Fig. 7, a and b).
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MM patients have been reported to have defects in their T cells
such as increased susceptibility to apoptosis (reviewed in Ref.
25), which may make observations on normal T cells less
valid. Therefore, peripheral blood T cells from patients in plateau
phase of MM were pretreated with IL-2 or IL-15 as before, and
restimulated in the presence of U266 cells. As with normal T cells,
IL-15-treated T cells were not inhibited in their proliferation,
whereas IL-2-treated T cells were significantly inhibited (Fig. 8a).
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b). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is an immune escape mechanism used by
such diverse tumors as breast carcinoma, renal cell carcinoma,
Hodgkin’s disease, and MM (1, 2, 3, 4, 5). The effects that TGF
has on the IL-2-induced phosphorylation of STAT3 and STAT5 have been
subject to conflicting reports. Here we have shown that TGF from MM
tumor cells is responsible for inhibition of both STAT3 and STAT5
phosphorylation in response to IL-2, and that only STAT3 appears to be
necessary for T cells to maintain their proliferative capacity. The
suppression of intracellular signaling and proliferation mediated by
TGF could be completely overcome by treating T cells with IL-15.
Moreover, IL-15 treatment could restore the ability of T cells to
resist TGF-mediated suppression of IL-2 responses.
It is well documented that MM cells and TGF interrupt T
cell progression into the cell cycle and autocrine IL-2 pathway, and
that T cells cannot respond to exogenous IL-2 (1, 6, 7, 8, 19). The exact mechanism of TGF-induced suppression of IL-2
responses is controversial. One murine study reported an inhibition of
STAT5 phosphorylation (6) (although this study used
TGF2), whereas another found that both STAT3 and STAT5 were
inhibited (7). In a study of human T cells it was reported
that STAT5 was unaffected by TGF1 (8). Our results
here, using a TGF1-producing tumor, show that both STAT3 and STAT5
phosphorylation in response to IL-2 are inhibited. The down-regulation
of both STAT3 and STAT5 phosphorylation by MM cells was directly
attributable to TGF production by the tumors as treatment with LAP
restored their phosphorylation, in accordance with our previous
findings on T cell proliferation (1). It is interesting to
note that our results agree with the (murine) observations of Han et
al. (7), who also used natural rather than rTGF. TGF
is produced as an inactive promolecule (conjugated to LAP, hence LAP is
an excellent TGF-specific blocking agent), which is enzymatically
cleaved when released from the cell (20). rTGF requires
artificial cleavage to form an active molecule. The rTGF1 used in
these experiments was cleaved by acidification of the culture medium
containing the recombinant protein (manufacturer’s data). This may
alter its secondary structures and could influence the apparently
different effects on STAT signaling between natural and rTGF.
Signaling through CD25 appears to be critical for suppression
of STAT3 phosphorylation by tumor cells. The CD25-BDB2
cells phosphorylated STAT3 in response to IL-2, irrespective of the
presence of MM cells, whereas in CD25+ cells
STAT3 phosphorylation was completely inhibited and the proliferation of
the cells was greatly reduced. This apparent role for STAT3 in
proliferation may explain previous findings that inhibition of STAT5
phosphorylation does not reduce IL-2-induced proliferation in human T
cells (21). This is further confirmed by the finding that
tumor cells inhibited STAT5 phosphorylation in BDB2 cells although this
was insufficient to reduce proliferation of this cell line in response
to IL-2. STAT5 is thought to be responsible for up-regulation of CD25
and IL-2 production (22), and as BDB2 cell lines do not
express either of these factors, this may explain why down-regulation
of STAT5 phosphorylation has no effect on these cells. Also, human
cells use IL-2-induced STAT5 phosphorylation as an anti-apoptotic
rather than proliferative signal (23) (in direct contrast
to mice, Ref. 24) again perhaps explaining why its
inhibition does not affect normal T cell or BDB2 proliferation.
Finally, the BDB2 cell line demonstrates that the
IL-2/IL-15c intermediate affinity receptor
is capable of signaling through both STAT3 and STAT5 in response to
IL-2, and to our knowledge this is a novel finding in human cells. It
is well established that the intermediate affinity receptor can induce
IL-2-dependent proliferation (24), but the signaling
differences were unelucidated. MM cells inhibit STAT3 and STAT5 in high
affinity receptor-bearing cells, but STAT5 not STAT3 in BDB2 cells.
Thus signaling in BDB2 cells appears to be differentially controlled
compared with high-affinity receptor-bearing cells. Intermediate
affinity receptors can dimerize in response to IL-2, but only CD25 can
make them oligomerize, a factor thought to account for the differences
in IL-2 response (25). The results shown here suggest that
there may be differences in recruitment or activation of signal
transduction molecules, and this will require further
investigation.
On the basis that T cells are deficient in their responses to
IL-2 when cocultured with MM cells, and that this deficiency was
strongly associated with signaling through CD25 (i.e.,
CD25- cells were not suppressed), we examined
the role of IL-15 in maintaining T cell activation. Using IL-15, we
were able to restore T cell-proliferative and activation responses in
the presence of MM cells. In primary activation of resting T cells with
mitogen, IL-15 restored proliferation and allowed progression through
the autocrine IL-2 pathway, based on CD25/CD69 expression. Thus IL-15
overcame the "block" that MM cells place on activation of resting T
cells. This is almost certainly due to the ability of IL-15 to drive T
cells into the autocrine IL-2 pathway (15). In addition,
we found that IL-15-treated T cells were more sensitive to IL-2 than
IL-2-treated cells, a previously unreported finding. It is possible
that as IL-15 heightens IL-2 sensitivity, T cells are far more able to
progress through the autocrine IL-2 pathway even in the face of the
TGF-producing MM cells, which are lowering their capacity to make
IL-2 (6).
rIL-15 was protective at a standard dose of 10 ng/ml, but
"physiological" levels of natural cytokine were also effective.
Monocytes treated with SAC (or LPS)/IFN- produce IL-15
(13), and such cells were capable of driving T cells
through normal activation in the presence of MM cells, whereas
untreated monocytes did not. Activated monocytes produce other
proinflammatory mediators such as IL-12 (26); however, the
protective effect was completely attributable to IL-15, as sIL-15R
blocked any rescue.
When the capacity of activated T cells to proliferate to IL-2
or IL-15 in the presence of MM cells or rTGF1 was assessed,
IL-15-treated cells were completely unaffected, whereas T cells were
unable to respond to IL-2. Indeed, rTGF1 appeared to be synergistic
with IL-15 in driving T cell proliferation. IL-15-treated cells’
phosphorylation of STAT3 and STAT5 was identical with or without tumor
cells present, whereas IL-2-treated cells were unable to phosphorylate
STAT3 or STAT5. Thus, when IL-15 protects T cells from MM-induced
inhibition the cytokine maintains STAT3 and STAT5 phosphorylation. The
finding that IL-15-induced proliferation is not inhibited by TGF is
novel. TGF has been reported to inhibit IL-15-induced IFN-
production in T cells (27), but the intracellular events
that control this have yet to be elucidated. As induction of IFN- by
IL-12 (8) is STAT4 mediated, and TGF does not
apparently affect phosphorylation of STAT4 (8), it
is possible that TGF does not interfere with this mechanism in T
cells.
T cells pretreated with IL-15 responded with maximum proliferation to IL-2 concentrations one-tenth that required to achieve the same proliferative rates in IL-2-pretreated cells, indicating an increased longevity and sensitivity of IL-2 response. This translated into an ability to proliferate and phosphorylate STAT3 in the presence of tumor cells. Significantly, T cells from MM patients could also respond to IL-2 in the presence of MM cells when pretreated with IL-15. IL-15 induces similar CD25 expression levels to IL-2, but may increase IL-2 production by T cells (25). Therefore, the increased sensitivity of T cells to IL-2 seen here may be due to increased endogenous IL-2 expression by the T cells, resulting in a magnified response to IL-2, as the rate of proliferation in the autocrine IL-2 pathway is dependent on the total amount of IL-2 available to T cells (28). This explanation may be at odds with the finding that adding increased doses of IL-2 does not overcome tumor suppression, and may indicate that another mechanism is in operation.
In the setting of tumor and HIV immunotherapy, IL-15 provides
hope that where T cell responses to IL-2 are inefficient or defective
they can be reactivated (29, 30). However, administration
of IL-15 to patients is potentially risky, as IL-15 is implicated in
the pathogenesis of autoimmune diseases such as rheumatoid arthritis
(31), and in the case of MM, potentially could act as a
tumor cell survival factor (32, 33). Therefore, the
finding here that IL-15-treated T cells regained the ability to respond
to IL-2 in the presence of tumor cells is significant. It raises the
possibility that T cells treated ex vivo with IL-15 could be
administered to patients and retain responses to IL-2, which can be
administered clinically. As the production of TGF by tumor cells is
a common means of immune suppression, the findings of this study may be
applicable to T cell therapies in a variety of malignancies.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John Campbell, Academic Transfusion Medicine Unit, Department of Medicine, University of Glasgow, Royal Infirmary, Glasgow, G31 2ER, U.K. E-mail address: jdmc1v{at}clinmed.gla.ac.uk
3 Abbreviations used in this paper: MM, multiple myeloma; LAP, latency-associated peptide; SEB, staphylococcal enterotoxin B; SAC, "Pansorbin" Staphylococcus aureus cells; Jak, Janus kinase; h, human; BM, bone marrow; s, soluble.
Received for publication November 14, 2000. Accepted for publication April 19, 2001.
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  L. B. Baughn, M. Di Liberto, R. Niesvizky, H. J. Cho, D. Jayabalan, J. Lane, F. Liu, and S. Chen-Kiang CDK2 Phosphorylation of Smad2 Disrupts TGF-{beta} Transcriptional Regulation in Resistant Primary Bone Marrow Myeloma Cells J. Immunol., February 15, 2009; 182(4): 1810 - 1817. [Abstract] [Full Text] [PDF]
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 T. Yamashita, H. Tamura, C. Satoh, E. Shinya, H. Takahashi, L. Chen, A. Kondo, T. Tsuji, K. Dan, and K. Ogata Functional B7.2 and B7-H2 Molecules on Myeloma Cells Are Associated with a Growth Advantage Clin. Cancer Res., February 1, 2009; 15(3): 770 - 777. [Abstract] [Full Text] [PDF]
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 K. Wendt, E. Wilk, S. Buyny, J. Buer, R. E. Schmidt, and R. Jacobs Gene and protein characteristics reflect functional diversity of CD56dim and CD56bright NK cells J. Leukoc. Biol., December 1, 2006; 80(6): 1529 - 1541. [Abstract] [Full Text] [PDF]
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 P. J. Lucas, S.-J. Kim, C. L. Mackall, W. G. Telford, Y.-W. Chu, F. T. Hakim, and R. E. Gress Dysregulation of IL-15-mediated T-cell homeostasis in TGF-beta dominant-negative receptor transgenic mice Blood, October 15, 2006; 108(8): 2789 - 2795. [Abstract] [Full Text] [PDF]
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O. Goodyear, K. Piper, N. Khan, J. Starczynski, P. Mahendra, G. Pratt, and P. Moss CD8+T cells specific for cancer germline gene antigens are found in many patients with multiple myeloma, and their frequency correlates with disease burden Blood, December 15, 2005; 106(13): 4217 - 4224. [Abstract] [Full Text] [PDF]
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P. Leveen, M. Carlsen, A. Makowska, S. Oddsson, J. Larsson, M.-J. Goumans, C. M. Cilio, and S. Karlsson TGF-{beta} type II receptor-deficient thymocytes develop normally but demonstrate increased CD8+ proliferation in vivo Blood, December 15, 2005; 106(13): 4234 - 4240. [Abstract] [Full Text] [PDF]
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I.-K. Park, L. D. Shultz, J. J. Letterio, and J. D. Gorham TGF-{beta}1 Inhibits T-bet Induction by IFN-{gamma} in Murine CD4+ T Cells through the Protein Tyrosine Phosphatase Src Homology Region 2 Domain-Containing Phosphatase-1 J. Immunol., November 1, 2005; 175(9): 5666 - 5674. [Abstract] [Full Text] [PDF]
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S. C. McKarns and R. H. Schwartz Distinct Effects of TGF-{beta}1 on CD4+ and CD8+ T Cell Survival, Division, and IL-2 Production: A Role for T Cell Intrinsic Smad3 J. Immunol., February 15, 2005; 174(4): 2071 - 2083. [Abstract] [Full Text] [PDF]
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B. H. Nelson, T. P. Martyak, L. J. Thompson, J. J. Moon, and T. Wang Uncoupling of Promitogenic and Antiapoptotic Functions of IL-2 by Smad-Dependent TGF-{beta} Signaling J. Immunol., June 1, 2003; 170(11): 5563 - 5570. [Abstract] [Full Text] [PDF]
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 A. Mookerjee, P. C. Sen, and A. C. Ghose Immunosuppression in Hamsters with Progressive Visceral Leishmaniasis Is Associated with an Impairment of Protein Kinase C Activity in Their Lymphocytes That Can Be Partially Reversed by Okadaic Acid or Anti-Transforming Growth Factor {beta} Antibody Infect. Immun., May 1, 2003; 71(5): 2439 - 2446. [Abstract] [Full Text] [PDF]
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 F. Annunziato, L. Cosmi, F. Liotta, E. Lazzeri, R. Manetti, V. Vanini, P. Romagnani, E. Maggi, and S. Romagnani Phenotype, Localization, and Mechanism of Suppression of CD4+CD25+ Human Thymocytes J. Exp. Med., August 5, 2002; 196(3): 379 - 387. [Abstract] [Full Text] [PDF]
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