| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Departments of
*
Cell Biology and
Pathology, New York University School of Medicine, New York, NY 10016
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
is
secreted. However, on conjugate formation between nonlytic TIL and
cognate tumor cells in vitro, the microtubule-organizing center (MTOC)
does not localize to the immunological synapse, thereby precluding
exocytosis of preformed lytic granules and accounting for defective TIL
lytic function. Recovery of TCR-mediated, activation-dependent MTOC
mobilization and lytic activity requires proteasome function, implying
the existence of an inhibitor of MTOC mobilization. Our findings show
that the regulated release of TIL cytolytic granules is defective
despite functional TCR-mediated signal
transduction. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Human antitumor immune responses do not usually correlate with tumor remission but has motivated considerable research in cancer immunotherapy. Although a variety of protocols have proved successful in immunization of rodents to resist primary tumor challenges, only rarely have large preexisting tumors been eradicated (6). The failure to cure established rodent tumors and the modest success of human experimental immunotherapies, together with the realization that antigenic human cancers elicit immune response but are usually not eliminated, has sparked interest in understanding the basis for inadequate antitumor T cell immune responses (7).
The presence of TIL in human cancers demonstrates that tumor Ags are expressed in situ that are capable of eliciting antitumor T cell immune response, which nonetheless fails to eliminate tumors (8). The lytic function of CD8+ TIL has been investigated in various human (9) and murine (10, 11) tumors and found to be defective. Deficient lytic function is transient because, on purification from tumor cells and culture in vitro, tumor-specific killing can be detected (12).
We have investigated the basis for defective TIL lytic function in transplantable murine tumor models. Freshly isolated TIL, although nonlytic in vitro, contain mRNAs encoding effector phase cytokines, serine esterases, and perforin characteristic of cytolytic effector T cells. Furthermore, perforin and granzyme B proteins are synthesized and packaged into lytic granules showing that TIL are mature CD8+ T cells. TIL recover perforin-mediated, Ag-specific lysis if purified and cultured briefly in vitro. After recovery of lytic function, on conjugate formation with cognate tumor cells in vitro, TIL mobilize cytotoxic granules to the immunological synapse, whereas granules in freshly isolated, nonlytic TIL do not migrate. In addition, the microtubule-organizing center (MTOC) does not localize to the immunological synapse in nonlytic TIL. This defect precludes movement of lytic granules to the immunological synapse and accounts for defective TIL lysis. Biochemical analyses demonstrated that TCR kinase-mediated signaling is functional in freshly isolated, nonlytic TIL. Our findings show that after arrival to the tumor microenvironment, antitumor T cells are unable to release cytolytic granules on contact with tumor which may permit tumor escape from T cell lysis.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Male C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were housed four per cage in a barrier facility and maintained on a 12-h light/dark cycle (7 a.m. to 7 p.m.) with ad libitum access to food and water. A sentinel program revealed that the mice were murine hepatitis virus negative and the tumor cell lines are murine hepatitis virus negative. Experiments involving animals were conducted with the approval of the New York University School of Medicine Committee on Animal Research.
Tumors
MCA-38 adenocarcinoma (13), P-815 cells (14), and 6-1 fibrosarcoma (15) have been described recently (12). Then 1–2 x 106 cells were injected s.c in 0.1 ml HBSS for tumor induction.
Tissue culture
Sources and use of all tissue culture reagents have been described recently (12).
Isolation of TIL
Isolation of TIL from tumors by immunomagnetic separation was recently described (12). Potentially inhibitory effects of the immunobead isolation protocol on TIL lytic function are unlikely because we have reisolated TIL with anti-CD8 magnetic beads after recovery in vitro, and those cells are lytic. As a further control, we have prepared CTL by primary MLR in vitro followed by isolation with anti-CD8 immunobeads. These cells are fully lytic, again showing that the isolation protocol is without effect on lytic activity.
Flow cytometric intracellular staining of T cells
Single-cell suspensions were prepared from tumors, primary MLR, or spleens of control mice by enzyme digestion and magnetic immunobeading as recently described (12). For granzyme B analysis from all sources, T cells were isolated by positive selection using anti-CD8-conjugated beads. For analysis of perforin expression in TIL, positive selection was also used. For granzyme B analysis of splenocytes, T cells were prepared by negative selection using anti-MAC1-, anti-CD4-, and anti-B220-conjugated beads. Flow cytometric analyses were performed as recently described (12). Primary Abs (either species- and isotype-matched nonimmune Ig, rat anti-perforin (clone KM585; Kamiya Biochemical, Seattle, WA), goat anti-granzyme B (Santa Cruz Biotechnology, Santa Cruz, CA)) were used at 0.5 µg/106 cells followed by appropriate secondary Abs (goat anti-rat Ig (Caltag Laboratories, Burlingame, CA) or donkey anti-goat (Jackson ImmunoResearch Laboratories, West Grove, PA)).
Generation of MLR primary CTL in vitro
CTL were prepared from primary MLR as described (12).
Chromium release assay
CTL activity was determined in standard
51Cr release assays as recently described
(12). Redirected cytolysis assays, using anti-CD3
Ab 145-2C11 (at 0.001 mg/ml final) and P-815 cells as targets, were
performed as described (16). Hamster IgG was used as
control Ab for redirected assay, and lysis of labeled targets in the
presence of effector cells plus control IgG was equivalent to
spontaneous release. Specific lysis was determined as described
(12).
Pharmacological inhibition of T cell kinases before lysis assay used
wortmannin, bisindolmaleamide, or PD98059 (all obtained from
Calbiochem, La Jolla, CA) at the dosages indicated in Fig. 6
.
|
CD8+ TIL (6 x
105) were mixed with tumor cells (3 x
105) in 0.6 ml serum-free RPMI 1640 at room
temperature. Cells were centrifuged at 170 x g for 10
min to promote conjugate formation, incubated for 5–10 min at 37°C,
and transferred to coverslips (VWR Scientific, South Plainfield, NJ)
pretreated with 1% Alcian blue. Cells were fixed in 4%
paraformaldehyde for 30 min at room temperature, permeabilized with
0.01% saponin, 0.5% BSA in PBS (30 min at room temperature), and then
then stained with 0.5 µg/ml of mouse monoclonal anti-
-tubulin
Ab (Boehringer Mannheim, Indianapolis, IN), mouse anti-Rab 27a (BD
Transduction Laboratories, San Diego, CA), rabbit anti-PYK-2 (a
gift from J. Schlessinger, New York University School of Medicine), or
goat polyclonal anti-granzyme B Ab (Santa Cruz Biochemicals, Santa
Cruz, CA) (1 h at 37°). After three washes (0.01% saponin, 0.5% BSA
in PBS), cells were incubated with secondary Ab (FITC-conjugated
anti-mouse IgG for -tubulin and Rab 27a or PE- or
FITC-conjugated anti-goat for granzyme B) (30 min at RT°), washed
three times (0.01% saponin, 0.5% BSA in PBS), incubated with 0.1%
Evans blue for 30 s, and washed thoroughly. Preparations were
covered with mounting solution before fluorescence or confocal
microscopy using a MRC 600 device (Bio-Rad, Hercules, CA). MRC images
were converted to PICT files using mrc2 M software (version 1.9.3).
Immunoblot analysis
Activation of purified T cells in vitro and immunoblot analyses was performed as described (12). Some immunoblotting experiments used whole cell detergent extracts prepared from freshly isolated TIL (or TIL that were recovered in vitro before extract preparation) which were not activated in vitro.
TIL subcellular fractionation and PKC analysis was performed essentially as described (17). Equal cell equivalents of soluble and membrane fractions were subjected to SDS-PAGE followed by immunoblotting with isoform-specific Abs and detection with ECL (Amersham-Pharmacia Biotech, Little Chalfont, Buckinghamshire, England).
Antibodies used for immunoblotting were: mouse monoclonal
anti-phosphotyrosine, (clone PY20, Transduction Laboratories,
Lexington, KY), mouse monoclonal anti-ERK1 (clone MK1, Transduction
Laboratories), polyclonal rabbit anti-active MAPK (pTEpY, Promega,
Madison, WI), polyclonal rabbit anti-Akt and polyclonal rabbit
anti-phospho-Akt (Ser473) (Cell Signaling Technology, Beverly, MA),
mouse anti-Rab 27a, rabbit anti-PYK-2, and anti-PKC
isoforms 
, 
, or (Upstate Biotechnologies, Lake Placid, NY),
and (Transduction Laboratories).
Intracellular calcium mobilization studies
CD8+ TIL were isolated and resuspended at
106 cells/ml in serum-free RPMI 1640. Cells were
incubated with 1 µM Indo-1 (Sigma Chemical, St. Louis, MO) for 1
h, washed three times, and stimulated (10 µg/ml anti-CD3 mAb
(clone 500-A2) or 1 µM ionomycin), and the OD398 and
480 was measured over time. Excitation was at 340 nM in a
Fluoromax 2 spectrofluorometer calibrated as described
(18).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A). Freshly purified TIL are
not lytic in standard four hr assays, however, if TIL are cultured in
vitro in the presence of IL-2, substantial lytic activity is detected.
The level of lysis is comparable to that demonstrated by primary MLR
CTL at equivalent E:T ratios (Fig. 6
).
|
B) (TIL were also tested for lytic function by
redirected lysis assay (in which Ag specificity is irrelevant) and
found to be nonlytic showing that the lytic defect is not caused by an
unknown inhibitory MCA-38 tumor influence in vitro (data not shown)).
TIL isolated from a variety of other tumors were similarly tested for
Ag-specific lysis after recovery of lytic function in vitro and in all
cases TIL demonstrate tumor-specific lysis (fibrosarcoma 6-1
(15), EL4 thymoma (19), EG.7 (EL4 expressing
an MHC class I-restricted OVA peptide) (20), MC57G sarcoma
(21), and mastocytoma P815 (14); data not
shown).
The mechanism of cytolysis in recovered TIL was tested by inclusion of
either EGTA (which inhibits requisite functional polymerization of
perforin, MTOC mobilization, and lytic granule exocytosis by chelating
free calcium (22)), or blocking Ab to Fas ligand (FasL;
Fig. 1C). Anti-FasL Ab had no effect, however, EGTA
completely abrogated lysis showing that target cell killing was
perforin-mediated. In addition, the rapid kinetics of in vitro
cytolysis by recovered TIL is characteristic of perforin-, but not
FasL-, mediated lysis (23).
Collectively these experiments show that freshly isolated TIL are defective in tumor cell lysis in vitro but lytic function is restored after purification from tumor and brief culture in vitro. In addition, TIL lysis of cognate tumor cells is Ag-specific and perforin-mediated.
Recovery of TIL lytic function
The recovery of TIL lytic function was examined. In preliminary
experiments TIL were incubated in various concentrations of rIL-2 for
different periods of time and cytolysis determined. As little as 5 U/ml
of rIL-2 for 12 h was sufficient to permit maximal recovery of
lytic function (data not shown). Furthermore, TIL could recover
significant lytic function if cultured in vitro for 6 h without
additional medium supplements, typically 50–75% of maximal lysis
(Fig. 2). Actinomycin D, cycloheximide,
or brefeldin A had no effect on recovery of cytolytic function if
included during the 6 h recovery period showing that recovery of
lytic function does not require RNA, protein synthesis, or protein
secretion. Recovery of maximal lytic activity required 
12 h of in
vitro culture, both RNA and protein synthesis (data not shown), and
exogenous IL-2.
|
One potential reason for the inability of TIL to lyse tumor target
cells in vitro may be related to the state of TIL maturation which is
reflected in the transcriptional activity of effector phase genes
(24). The observation that protein or RNA synthesis is not
required for recovery of significant TIL lytic function suggested that
TIL are mature effector T cells. We characterized TIL further by
analyzing expression of genes characteristic of the effector phase.
CD8+ TIL were purified from tumors and used to
prepare cDNA for programming of RT-PCR analysis of cytokine gene
transcripts. Freshly isolated CD8+ TIL contain
mRNAs encoding IL-2, IFN-, perforin, granzyme A, and granzyme B
(12).
In addition, expression of granzyme B and perforin proteins in TIL was
examined (Fig. 3A). As
anticipated, primary MLR CD8+ T cells
demonstrating high levels of lysis in vitro (compare Fig. 6) contain
both granzyme B and perforin. Freshly isolated, nonlytic TIL contain
equivalent levels of perforin and slightly higher levels of granzyme B
compared with MLR T cells. The level of perforin protein in TIL does
not change when TIL are cultured in vitro for 12 h (data not
shown) suggesting that recovery of lytic function is not due to
increased expression of perforin.
|
B)
and in purified TIL (data not shown), and found that perforin and
granzyme B (Fig. 5A) proteins are localized in cytoplasmic
granules.
|
TIL cytokine secretion
The RNA analysis showed that TIL contain cytokine mRNAs
(12) but did not reveal whether these cytokines are
secreted. To address this, purified nonlytic TIL were stimulated in
vitro and secretion of IL-2 and IFN- assessed by ELISA (Fig. 4). Secretion of IFN- was rapid: after
2 h of in vitro activation 300 U/106
cells were produced. After 4 h of activation, high levels of
IFN- were produced, 3000 U/106 cells.
|
For effector T cells to demonstrate lytic function in response to
TCR-mediated signaling after target cell recognition, preformed
cytotoxic granules must accumulate under the plasma membrane closely
apposed to target cells, fuse to the membrane, and then release granule
contents. Because TIL contain perforin and serine esterases but are
nonlytic, we considered the possibility that granule exocytosis was
defective. We tested whether localization of lytic granules to the
immunological synapse occurred on TCR-mediated recognition of target
cells by redirected assay in vitro (Fig. 5A). TIL, either freshly
isolated or after in vitro recovery, were mixed with target cells for
5–10 min, and then the intracellular localization of lytic granules
was determined by immunofluorescence microscopy using Ab to granzyme B.
In freshly isolated, nonlytic TIL, only 5% of T cells forming
conjugates orient lytic granules to the area of tumor cell contact.
However, in 60–70% of conjugates formed between tumor cells and
recovered TIL, granzyme B+ granules were
reoriented to the immunological synapse. Representative pictures of
granzyme B staining are shown in Fig. 5A, where in recovered
but not freshly isolated TIL, lytic granules are easily visualized in
close apposition with tumor cell targets.
Exocytosis of TIL lytic granules is blocked due to failure of the MTOC to localize to the T cell immunological synapse
After TCR-mediated recognition of cognate Ag, migration of lytic
granules to the immunological synapse requires several sequential
biochemical events including orientation of the MTOC to the T cell
immunological synapse. The MTOC is a multicomponent structure that is
the site for aggregation of cytoplasmic microtubules on which lytic
granules migrate to the immunological synapse, where they fuse to the
plasma membrane to release lytic enzymes. Because TIL were determined
to contain mature lytic granules and because we showed that
mobilization of granules to the T cell immunological synapse was
defective, we asked whether TIL MTOC localized to the area of cell
contact on formation of conjugates with cognate tumor target cells. As
was found by analysis of lytic granule migration, TIL MTOC did not
localize to the immunological synapse (Fig. 5B). In TIL, the
lytic function of is restored by purification and short term in vitro
culture, the MTOC localizes to the immunological synapse within 5–10
min of contact with cognate tumor in vitro. Granule migration in
recovered TIL does not occur if noncognate tumor is used as target
cells (data not shown).
This finding shows that nonlytic, freshly isolated TIL are defective in an obligate early step in the process of TCR-mediated release of preformed cytotoxic granules, that of MTOC mobilization. Furthermore, recovery of lytic function is correlated with MTOC reorientation to the immunological synapse.
Conjugate formation by redirected assay does not restore lytic function or lytic granule mobilization
Freshly isolated, nonlytic TIL were observed to form fewer
conjugates with cognate target cells in vitro compared with TIL after
recovery (Table I). One possible
interpretation of this finding is that target cell contact by TIL may
be unstable, which might result in insufficient activation of T cell
lytic function. We consider this interpretation unlikely: when
conjugates are formed between nonlytic TIL and target cells by bridging
TIL and target cells with anti-CD3 Ab (redirected assay), an
equal number of conjugates are formed by nonlytic TIL as for lytic TIL,
but cytolysis still does not occur (Table I). This finding shows that
target cell conjugate formation is necessary but insufficient for
cytolysis. Recovered TIL in the redirected conjugate formation assay
are both lytic, and their lytic granules mobilize to the immunological
synapse (Fig. 5A), but freshly isolated TIL are nonlytic and
do not mobilize their lytic granules. Collectively, these observations
show that even when nonlytic TIL form conjugates at the same frequency
as lytic TIL, lytic granules do not localize to the immunological
synapse.
|
Release of granules from cytolytic cells involves a Ras-dependent
and a Ras-independent pathway of signal transduction. NK cells
activated to release granules, either on recognition of MHC class
I-negative target cells or via FcRIII ligation, use signal
transduction involving MAPK-ERK, Vav, Pyk-2, PI3 kinase, and Syk 70
kinases (25). Granule release in T cells is less well
understood: one report shows that MAPK-ERK activation is required,
thought to be activated primarily through the TCR pathway
(26); whereas other reports implicate PI3 kinase
(27) or PKC in lytic function (28). After
recovery of TIL lytic function, the role of three major T cell protein
tyrosine kinases in TIL lysis was investigated by inclusion of various
inhibitors during lysis assays. For these experiments, the redirected
assay was used, so that direct comparison of TIL function with primary
MLR CTL T cells (used as positive controls) could be made under
identical conditions. Because the results of lysis assays were
consistent when conducted at different E:T ratios, for comparison of
the effects of different inhibitors, only data from a single E:T ratio
(10:1) is shown. Over a range of concentrations typically used in
inhibition assays (27) inhibition of either PI3 kinase
(wortmannin), ERK (PD98059), or PKC (bisindolmaleamide) caused
dose-dependent reduction of lytic function (Fig. 6). MLR CTL lytic function was also
inhibited in a manner similar to that for TIL, showing that the
requirement for PI3 kinase, ERK, and PKC in target cell lysis is not
restricted to TIL.
Proximal TCR-mediated signal transduction is normal in nonlytic TIL
Using either NK cell or T cell lines, others have shown that
pharmacological inhibition of ERK (29), PKC
(28), or PI3 kinase (30) blocks lytic
function. We analyzed TIL for the presence and activation status of
those kinases suggested to play a role in granule release. First, we
examined TIL for the pattern of total tyrosine-phosphorylated proteins
by immunoblot analysis of lysates prepared from freshly isolated TIL
before and after activation in vitro with anti-TCR Ab. As a
control, spleen CD8+ T cells prepared by
identical procedures were used for comparison. Similar to spleen cells,
TIL have a very low basal level of tyrosine-phosphorylated proteins
and, on activation in vitro, several prominent protein species are
rapidly phosphorylated (Fig. 7A) including ZAP 70 and
mitogen-activated protein/extracellular signal-related kinase kinase
(data not shown). This observation shows that TIL proteins are not
hyperphosphorylated due to overstimulation in the tumor
microenvironment, which might inhibit subsequent activation events, and
also that TCR signal transduction is intact at least at the level of
the proximal components of the pathway.
|
A). Stripping the
immunoblot membrane and reprobing with Ab specific for activated
(phosphorylated) ERK revealed enhanced phosphorylation of the p42 form
of ERK (ERK2) in TIL, but not ERK1, in contrast to activated spleen T
cells which contain both ERK1 and ERK2.
On discovery that TIL do not contain p44 phospho-ERK, we considered
that the failure to activate both forms of ERK may be the biochemical
basis for defective MTOC orientation and lytic function. This
possibility was further investigated by assessment of the levels of ERK
proteins in TIL whose lytic function was restored. Lysates were
prepared from either freshly isolated or recovered TIL and, as positive
control, purified CD8+ T cells from primary MLR
cultures, and analyzed by immunoblotting. Lysates were prepared
directly after purification or following activation of TIL in vitro
with anti-TCR Ab. Aliquots of both recovered TIL and MLR cells were
determined to be lytic (40% lysis at E:T of 10:1- data not shown).
Nonlytic and lytic TIL and lytic MLR T cells have phospho-ERK2 but do
not contain activated p44 ERK demonstrating, we believe for the first
time, that ERK1 is not required for perforin-mediated lytic function in
T cells and eliminating the hypothesis that the failure to activate p44
ERK in TIL is the basis for lytic dysfunction.
Because activity of PI3 kinase has been shown to be involved in
cytolytic function of NK cells (30) and T cells (Fig. 6),
we asked whether PI3 kinase was activated in TIL. For this
determination we analyzed the level and activation status of the
immediate downstream target of PI3 kinase, Akt. Primary MLR
CD8+ T cells, again used as positive controls,
and freshly isolated TIL were prepared and either activated or not
before lysate preparation followed by immunoblot analysis. TIL contain
nonphosphorylated Akt protein which is rapidly phosphorylated on
activation showing that TCR-mediated signal transduction through PI3
kinase is intact in nonlytic TIL.
Because the inhibitor experiments implicated a role for PKC in CTL
function and also because of a previous report suggesting the same
requirement (28), activation of PKC was also tested. Cell
extracts were prepared either from freshly isolated or from TIL
recovered by in vitro culture. The subcellular localization of several
PKC isoforms was determined by fractionation of extracts into membrane
and cytosolic fractions that were subjected to SDS-PAGE and
immunoblotting with isoform-specific Abs (Fig. 7B). TIL
contain similar levels of membrane-associated PKC compared with control
T cells, except PKC- which has higher membrane-associated levels
thando control spleen cells. (Because both nonlytic or recovered TIL
have higher PKC- levels, this observation appears unrelated to lytic
status.) On activation in vitro, additional PKC protein becomes
membrane associated. Compared with spleen cells, the kinetics of
activation-induced PKC membrane association in TIL is slightly delayed:
by 10 min of stimulation, a portion of PKC in spleen T cells becomes
membrane associated; whereas a shift in localization does not occur in
TIL until after 10 min of activation in vitro. Because the kinetics of
membrane-association of PKC is identical for lytic and nonlytic TIL,
the delay in activation of PKC relative to primary spleen
CD8+ T cells cannot be directly related to
defective TIL lysis. This analysis shows that there is no dramatic
difference between nonlytic and lytic TIL in activation of (several
isoforms of) the third protein tyrosine kinase the function of which is
suggested to be required for lytic activity, PKC.
TIL calcium flux is normal
Because TCR activation-induced release of lytic granules has been
shown to be dependent on calcium flux (31), we tested TIL
calcium mobilization. Although freshly isolated TIL are nonlytic,
calcium flux in response to activation was indistinguishable in
comparison with lytic TIL (Fig. 8).
Supporting this observation, treatment of nonlytic TIL with PMA plus
ionomycin did not restore lytic function (data not shown), showing that
although requirement for activation of the proximal signaling pathway
was circumvented, TIL lytic function may require another activating
event or may be blocked. Because rapid calcium flux requires
TCR-dependent signal transduction, this finding further supports the
notion that proximal TCR-mediated signaling is intact in TIL.
|
TIL localization of PYK-2 or Rab 27a
Membrane translocation of the nonreceptor tyrosine kinase PYK-2 has been recently shown to be essential for NK cell cytolysis (32). We compared nonlytic and lytic TIL for levels of PYK-2 protein (by immunoblotting) and also for PYK-2 subcellular localization on conjugate formation in vitro (by confocal microscopy). We found both lytic and nonlytic TIL have equivalent levels of PYK-2 protein, suggesting that down-regulation of PYK-2 expression does not contribute to the TIL lytic defect. Furthermore, and in contrast to PYK-2 function in NK cells, PYK-2 does not localize to the immunological synapse in fully lytic T cells, thereby excluding a potential PYK-2 defect in nonlytic TIL (data not shown).
Additionally, the intracellular movement of vesicles to the plasma
membrane during activation-induced lysis has been recently shown to be
dependent on the function of a small GTPase, Rab 27a (33, 34). Therefore, we examined nonlytic and lytic TIL for levels
(Fig. 9A) and subcellular
localization of Rab 27a (Fig. 9B). The level of Rab 27a
protein is identical in lytic and nonlytic TIL (Fig. 9A),
eliminating the possibility that nonlytic TIL do not produce sufficient
Rab 27a (or degrade it abnormally), thereby inhibiting lytic function.
Also, Rab 27a localization was investigated by confocal microscopy
(Fig. 9B). Lytic and nonlytic TIL were labeled with both
anti-Rab 27a (green) and anti-granzyme B (red) to visualize
colocalization of Rab 27a with lytic granules. Rab 27a protein is
distributed widely in T cells, but granzyme B+
lytic granules colocalize with anti-Rab 27a (yellow in Fig. 9B), showing that nonlytic TIL contain Rab 27a protein which
colocalizes with lytic granules albeit, as shown previously, not at the
immunological synapse.
|
Two types of data suggest that acquisition of the lytic
dysfunction in TIL may involve induction of a repressor: signal
transduction is functional in nonlytic TIL (and the levels and
localization of other components of the lytic machinery are normal
(PYK-2 and Rab 27a)) and recovery of lytic function does not require
protein synthesis. According to this notion, a putative repressor is
degraded during TIL recovery in vitro. This was directly tested by
performing recovery of TIL function in the presence or absence of two
different inhibitors of proteasome function, lactacystin and MG132
(Fig. 10). If proteasome inhibitors are
included during the 6-h incubation in vitro, there is a dose-dependent
inhibition of recovery of lytic function. However, if TIL are cultured
in vitro in the absence of inhibitor treatment, full lytic activity is
recovered, and addition of proteasome inhibitors after recovery is
without effect on lytic function.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We have analyzed the nature of the TIL lytic defect in murine tumors. After purification and brief culture in vitro, TIL demonstrate substantial tumor-specific, perforin-mediated cytolysis showing that TIL are transiently inhibited in lytic function. In keeping with the activated phenotype demonstrated by expression of various cell surface markers, TIL express high levels of FasL (12). However, after isolation and recovery in vitro, tumor cell lysis is exclusively due to exocytosis of lytic granules. FasL-mediated killing does not appear to be operative in tumors, because tumor cells are not detectably apoptotic in situ (12).
We have investigated a variety of considerations for defective TIL lysis including: TIL are immature (i.e., lacking expression of perforin/serine esterases); packaging of serine esterases into lytic granules is defective; or TIL are defective in granule exocytosis. Importantly, TIL contain high levels of perforin and serine esterase proteins that are packaged into lytic granules. These findings argue against the possibility that, after homing to the tumor site, TIL require further maturation defined by synthesis and packaging of serine esterases into granules.
TIL were considered to possibly resemble memory T cells in lymphoid organs which are usually nonlytic and require in vitro stimulation over many days to regain CTL activity (35). However, TIL recover lytic activity after only 6 h in vitro showing that their phenotype is distinct from memory cells. We further considered that TIL may resemble T cells that infiltrate normal nonlymphoid tissues in terms of their nonlytic phenotype. The best evidence in support of the notion that peripheral tissue T cells do not behave like TIL is provided by a recent report from Masopust et al. (36) showing that Ag-specific T cells can exist in an activated form in tissues and be directly lytic upon isolation. The observation that TIL are mature CTL that recover lytic function after removal from contact with cells within the tumor bed implies that the tumor microenvironment induces the lytic dysfunction. This supposition is supported by the observation that tumor-draining lymph node CD8+ CTL have a high level of tumor-specific lytic capability (data not shown) which is obviously lost on entrance to the tumor bed. Despite containing granules loaded with lytic enzymes, mobilization of the lytic granules to the immunological synapse is defective as shown in immunofluorescence microscopic analyses of TIL:tumor cell conjugates formed in vitro. Defective localization of lytic granules was further shown to be due to the inability to recruit the MTOC to the immunological synapse, an activation-dependent event that is required for the proper localization of granules and subsequent exocytosis and target cell lysis (24, 37, 38). Defective MTOC mobilization is not likely due to some inherent defect in expression of tumor Ags by tumor cells in situ, because lytic granules do not migrate when conjugates are formed between TIL and Ag-irrelevant P-815 cells by redirected assay. This finding suggests either that some signaling event required for MTOC function is lacking in TIL or that MTOC function is repressed in nonlytic TIL.
Signaling requirements for CTL degranulation have been studied by others where, on the basis of pharmacological inhibitor or Ag dependency studies, it was shown that TCR-mediated signaling is required for release of serine esterases (39). On recognition of cognate Ag, CTL activate the ras/MAPK/ERK pathway and therein the PI3 kinase pathway, both of which are apparently required for cytolytic function (31). Activated PKC has been shown to be required for lytic function in certain T cell clones by influencing MTOC mobilization to the immunological synapse (28). TIL have defective mobilization of the MTOC despite normal TCR-mediated calcium flux and PKC activation. Therefore, although there is a precedent in the literature describing a requirement for both calcium flux and PKC function in both MTOC function and T cell cytolysis, the specific defect in TIL must be downstream of calcium flux and PKC activation.
Pharmacological inhibitor studies have shown that ERK activation is required for lytic function in murine MLR CTL (40). Supporting the notion that ERK is involved in degranulation, Wei et al. (29) have shown that in vitro lytic function of human NK cell lines and freshly isolated large granular lymphocytes is dependent on both contact with cognate target cells and activation of ERK2, but not ERK1. Our experiments using TIL, or as controls CTL obtained from primary MLR cultures, show a similar requirement for ERK2 activation in lysis of cognate tumor cells. ERK protein is present in nonlytic TIL and exists in the nonphosphorylated form; on TCR ligation, TIL and MLR CTL rapidly activate p42 ERK2 but not p44 ERK1. The lack of detectable activated ERK2 in lytic T cells suggests that, although ERK1 is required for T cell development (41), the effector phase function of T cell killing uses only ERK2. These experiments show that TCR-mediated signaling in TIL is intact and the lytic defect in TIL is downstream of ERK2 activation.
Degranulation of human NK cells was shown to require activation of PYK-2 (32). We have examined the activation-dependent recruitment of PYK-2 to the immunological synapse and found, in contrast to that observed in NK cell degranulation, that PYK-2 does not associate with the immunological synapse in fully lytic TIL thereby excluding a defect in PYK-2 activation as the basis for the TIL lytic defect (data not shown).
After purification of TIL and brief culture, both localization of cytolytic granules to the immunological synapse and lytic activity are demonstrated. Recovery of TIL lytic function and granule localization does not require either RNA or protein synthesis, suggesting that acquisition of the lytic defect does not involve inactivation of a factor(s) required for granule mobilization. Rather, we suggest that a protein(s) is induced in TIL the expression of which prevents MTOC mobilization to the immunological synapse. This suggestion is further supported by our observation that inhibition of proteasome function during recovery in vitro blocks recovery of lytic function. Because TCR-mediated signal transduction is functional in nonlytic TIL, at least to the point of PI3 kinase-PKC-ERK activation (and therein calcium flux), a putative repressor of MTOC mobilization must act downstream of PI3 kinase-PKC-ERK activation. Such a repressor could act directly on the MTOC or may have an unknown target that is required for MTOC activation. Supporting this possibility is the observation that in a rat basophil cell line Ab to CD81 inhibits degranulation without inhibiting tyrosine phosphorylation or calcium mobilization (42), the phenotype demonstrated by nonlytic TIL. That precedent suggests that a similar regulatory mechanism may exist in TIL, a notion that we are at present pursuing.
|
Footnotes |
|---|
2 Abbreviations used in this paper: TIL, tumor-infiltrating lymphocyte; AICD, activation-induced cell death; MTOC, microtubule-organizing center; PKC, protein kinase C; FasL, Fas ligand; PI3 kinase, phosphatidylinositol 3-kinase.
Received for publication June 22, 2001. Accepted for publication August 22, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RI-mediated degranulation by CD81. J. Exp. Med. 186:1307.This article has been cited by other articles:
|
|
 |
|
  C. Barcia Jr, A. Gomez, J. M. Gallego-Sanchez, A. Perez-Valles, M. G. Castro, P. R. Lowenstein, C. Barcia Sr, and M.-T. Herrero Infiltrating CTLs in Human Glioblastoma Establish Immunological Synapses with Tumorigenic Cells Am. J. Pathol., August 1, 2009; 175(2): 786 - 798. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-C. Lin, L.-Y. Chang, C.-T. Huang, H.-M. Peng, A. Dutta, T.-C. Chen, C.-T. Yeh, and C.-Y. Lin Effector/Memory but Not Naive Regulatory T Cells Are Responsible for the Loss of Concomitant Tumor Immunity J. Immunol., May 15, 2009; 182(10): 6095 - 6104. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. O. Kilinc, T. Gu, J. L. Harden, L. P. Virtuoso, and N. K. Egilmez Central Role of Tumor-Associated CD8+ T Effector/Memory Cells in Restoring Systemic Antitumor Immunity J. Immunol., April 1, 2009; 182(7): 4217 - 4225. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Nonaka, M. Saio, T. Suwa, A. B. Frey, N. Umemura, H. Imai, G.-F. Ouyang, S. Osada, M. Balazs, R. Adany, et al. Skewing the Th cell phenotype toward Th1 alters the maturation of tumor-infiltrating mononuclear phagocytes J. Leukoc. Biol., September 1, 2008; 84(3): 679 - 688. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Wallace, V. Kapoor, J. Sun, P. Mrass, W. Weninger, D. F. Heitjan, C. June, L. R. Kaiser, L. E. Ling, and S. M. Albelda Transforming Growth Factor-{beta} Receptor Blockade Augments the Effectiveness of Adoptive T-Cell Therapy of Established Solid Cancers Clin. Cancer Res., June 15, 2008; 14(12): 3966 - 3974. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Monu and A. B. Frey Suppression of Proximal T Cell Receptor Signaling and Lytic Function in CD8+ Tumor-Infiltrating T Cells Cancer Res., December 1, 2007; 67(23): 11447 - 11454. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Y. Ma, N. Monu, D. T. Shen, I. Mecklenbrauker, N. Radoja, T. F. Haydar, M. Leitges, A. B. Frey, S. Vukmanovic, and S. Radoja Protein Kinase C{delta} Regulates Antigen Receptor-Induced Lytic Granule Polarization in Mouse CD8+ CTL J. Immunol., June 15, 2007; 178(12): 7814 - 7821. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. Hargadon, C. C. Brinkman, S. L. Sheasley-O'Neill, L. A. Nichols, T. N. J. Bullock, and V. H. Engelhard Incomplete Differentiation of Antigen-Specific CD8 T Cells in Tumor-Draining Lymph Nodes J. Immunol., November 1, 2006; 177(9): 6081 - 6090. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. E. Brown, C. Blank, J. Kline, A. K. Kacha, and T. F. Gajewski Homeostatic Proliferation as an Isolated Variable Reverses CD8+ T Cell Anergy and Promotes Tumor Rejection J. Immunol., October 1, 2006; 177(7): 4521 - 4529. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Meng, H. Harlin, J. P. O'Keefe, and T. F. Gajewski Induction of Cytotoxic Granules in Human Memory CD8+ T Cell Subsets Requires Cell Cycle Progression J. Immunol., August 1, 2006; 177(3): 1981 - 1987. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Koneru, N. Monu, D. Schaer, J. Barletta, and A. B. Frey Defective Adhesion in Tumor Infiltrating CD8+ T Cells J. Immunol., May 15, 2006; 176(10): 6103 - 6111. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. B. Frey and N. Monu Effector-phase tolerance: another mechanism of how cancer escapes antitumor immune response J. Leukoc. Biol., April 1, 2006; 79(4): 652 - 662. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. D. Kopcow, D. S. J. Allan, X. Chen, B. Rybalov, M. M. Andzelm, B. Ge, and J. L. Strominger Human decidual NK cells form immature activating synapses and are not cytotoxic PNAS, October 25, 2005; 102(43): 15563 - 15568. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Curtsinger, D. C. Lins, C. M. Johnson, and M. F. Mescher Signal 3 Tolerant CD8 T Cells Degranulate in Response to Antigen but Lack Granzyme B to Mediate Cytolysis J. Immunol., October 1, 2005; 175(7): 4392 - 4399. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Guleng, K. Tateishi, M. Ohta, F. Kanai, A. Jazag, H. Ijichi, Y. Tanaka, M. Washida, K. Morikane, Y. Fukushima, et al. Blockade of the Stromal Cell-Derived Factor-1/CXCR4 Axis Attenuates In vivo Tumor Growth by Inhibiting Angiogenesis in a Vascular Endothelial Growth Factor-Independent Manner Cancer Res., July 1, 2005; 65(13): 5864 - 5871. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Bronte, T. Kasic, G. Gri, K. Gallana, G. Borsellino, I. Marigo, L. Battistini, M. Iafrate, T. Prayer-Galetti, F. Pagano, et al. Boosting antitumor responses of T lymphocytes infiltrating human prostate cancers J. Exp. Med., April 18, 2005; 201(8): 1257 - 1268. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Schlereth, I. Fichtner, G. Lorenczewski, P. Kleindienst, K. Brischwein, A. da Silva, P. Kufer, R. Lutterbuese, I. Junghahn, S. Kasimir-Bauer, et al. Eradication of Tumors from a Human Colon Cancer Cell Line and from Ovarian Cancer Metastases in Immunodeficient Mice by a Single-Chain Ep-CAM-/CD3-Bispecific Antibody Construct Cancer Res., April 1, 2005; 65(7): 2882 - 2889. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Koneru, D. Schaer, N. Monu, A. Ayala, and A. B. Frey Defective Proximal TCR Signaling Inhibits CD8+ Tumor-Infiltrating Lymphocyte Lytic Function J. Immunol., February 15, 2005; 174(4): 1830 - 1840. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. P. O'Keefe, K. Blaine, M.-L. Alegre, and T. F. Gajewski Formation of a central supramolecular activation cluster is not required for activation of naive CD8+ T cells PNAS, June 22, 2004; 101(25): 9351 - 9356. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Chen, A. Purohit, E. Halilovic, S. J. Doxsey, and A. C. Newton Centrosomal Anchoring of Protein Kinase C {beta}II by Pericentrin Controls Microtubule Organization, Spindle Function, and Cytokinesis J. Biol. Chem., February 6, 2004; 279(6): 4829 - 4839. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Dobrzanski, J. B. Reome, J. A. Hollenbaugh, and R. W. Dutton Tc1 and Tc2 Effector Cell Therapy Elicit Long-Term Tumor Immunity by Contrasting Mechanisms That Result in Complementary Endogenous Type 1 Antitumor Responses J. Immunol., February 1, 2004; 172(3): 1380 - 1390. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Dobrzanski, J. B. Reome, J. A. Hollenbaugh, J. C. Hylind, and R. W. Dutton Effector Cell-Derived Lymphotoxin {alpha} and Fas Ligand, but not Perforin, Promote Tc1 and Tc2 Effector Cell-Mediated Tumor Therapy in Established Pulmonary Metastases Cancer Res., January 1, 2004; 64(1): 406 - 414. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. G. Carrabba, C. Castelli, M. J. Maeurer, P. Squarcina, A. Cova, L. Pilla, N. Renkvist, G. Parmiani, and L. Rivoltini Suboptimal Activation of CD8+ T Cells by Melanoma-derived Altered Peptide Ligands: Role of Melan-A/MART-1 Optimized Analogues Cancer Res., April 1, 2003; 63(7): 1560 - 1567. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Martin-Verdeaux, I. Pombo, B. Iannascoli, M. Roa, N. Varin-Blank, J. Rivera, and U. Blank Evidence of a role for Munc18-2 and microtubules in mast cell granule exocytosis J. Cell Sci., January 15, 2003; 116(2): 325 - 334. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Blohm, E. Roth, K. Brommer, T. Dumrese, F. M. Rosenthal, and H. Pircher Lack of Effector Cell Function and Altered Tetramer Binding of Tumor-Infiltrating Lymphocytes J. Immunol., November 15, 2002; 169(10): 5522 - 5530. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-J. Kim, R. R. Brutkiewicz, and H. E. Broxmeyer Role of 4-1BB (CD137) in the functional activation of cord blood CD28-CD8+ T cells Blood, October 16, 2002; 100(9): 3253 - 3260. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or cultured in medium containing 20 U/ml rIL-2 for
12 h before lysis assay (•). Target cells were cognate MCA-38
tumor cells in both assays. B, TIL exhibit Ag-specific
lysis. TIL were isolated and cultured overnight in the presence of
rIL-2 before cytolysis assays using either cognate MCA-38 tumor cells
(
), and additions were maintained during
the 4-h lysis assay. Isotype-matched nonimmune Ig has no effect on
lysis (data not shown).
