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*
Surgery Branch and
Department of Transfusion Medicine, Clinical Center, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In an attempt to reconcile the dichotomy observed between in vitro generated PBMC reactivity and in vivo tumor regression, we developed a sensitive molecular assay to directly detect specific low precursor CTL reactivity in bulk PBMC and in the local tumor microenvironment. In previous studies involving the immunization of cancer patients, monitoring of immune reactivity has involved single or multiple in vitro restimulation of PBMC to generate cultures with detectable reactivity (5, 6). More direct PBMC analysis with tetrameric HLA/peptide complexes (tHLA) has allowed for measurement of epitope-specific T cell precursor frequency (7) but has been limited by the sensitivity of FACS detection. Further, traditional tetramer analysis does not provide direct assessment of immunological reactivity of the identified cells. The local tumor site, the ultimate target of immunization, has rarely been examined serially in vaccine trials.
In this study, using molecular gene quantitation techniques, we report evidence of specific CTL reactivity in fresh cells obtained directly from the peripheral blood of patients immunized with the 209-2M peptide. We also demonstrate in vivo localization of vaccine-induced immune response by prospectively measuring therapy-specific changes within the tumor microenvironment from sequentially obtained fine needle aspirates of melanoma tumors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Each of the peptides utilized in this study was prepared according to Good Manufacturing Practice by Multiple Peptide Systems (San Diego, CA). The identity of each of the peptides was confirmed by mass spectral analysis. Peptide sequences are described below with their applications.
Cultured cell lines
The melanoma cell lines 624.38 Mel (HLA-A2+), 624.28 Mel (HLA-A2-), 888 (A2+) Mel (HLA-A2+), and 888 Mel (HLA-A2-) were established in the Surgery Branch, National Cancer Institute, and cultured as described (8).
Clinical protocols
All patients had histologically confirmed metastatic melanoma and had undergone a complete clinical evaluation including measurement and radiographic imaging of all evaluable tumor sites. HLA typing and subtyping for HLA class I was determined on patients’ PBL with sequence-specific primer-PCR. All patients provided informed consent before treatment and were verified to have been free of any treatment in the prior month, nor were they receiving immunosuppressive treatment including steroids. Before treatment, patients underwent leukapheresis. PBMC were isolated by Ficoll-Hypaque (ICN, Aurora, OH) separation and were cryopreserved at 108 cells/vial and stored at -180°C. In addition, patients underwent fine needle aspiration (FNA) of s.c. metastases immediately before treatment; sampling of the same lesion was repeated after 6–7 wk. Thus, because immunizations were administered at 3-wk intervals, PBMC and FNA were obtained simultaneously before treatment and 3–4 wk after the second vaccination. Aspirate material was examined and verified to contain melanoma cells at the bedside by a cytopathologist. The remainder of the aspirate was placed in ice cold culture medium (CM) consisting of RPMI 1640 (Biofluids, Rockville, MD) supplemented with 10 mM HEPES buffer, 100 U/ml penicillin-streptomycin (Biofluids), 0.03% L-glutamine (Biofluids), and 10% heat-inactivated FBS (Biofluids). The sample was immediately brought to the laboratory, centrifuged, placed in RNA lysis buffer (Qiagen, Santa Clarita, CA), and stored at -180°C until RNA isolation was performed.
Two sets of experiments were performed. First, a pilot study was done
to assess whether gene measurement techniques could provide information
comparable to the assessment of T cell reactivity obtainable with in
vitro sensitization experiments as previously described
(5). In this first study, vaccination-specific immune
reactivity in pre- and postimmunization PBMC was retrospectively
compared in 10 patients who had undergone vaccination with a
combination of four peptides including gp100, 209-2M (IMQVPFSV); gp100,
280-9V (YLEPGPVTV); MART-1 27–35 (AAGIGILTV), and tyrosinase 368–370D
(YMDGTMSQV). Each peptide (1 mg) was administered s.c. emulsified in
IFA (Montanide ISA-51, Seppic, France) at 3-wk intervals. Immune
reactivity against the 209-pa peptide (ITQVPFSV) (Table I
) was analyzed 3–4 wk after the second
immunization. Only 209-pa reactivity elicited by vaccination with
209-2M was assessed given that prior in vitro sensitization analysis
found no evidence of induction of PBMC reactivity toward the other
epitopes.
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Epitope-specific T cell staining with HLA-A2 tetramers
PE-tHLA complexes were synthesized as described previously (7). Recombinant HLA-A*0201 heavy chain containing a biotinylation site and recombinant ß2-microglobulin were synthesized and used for refolding of soluble HLA (sHLA) molecules in the presence of a HLA-A*0201 binding peptide. sHLA molecules were prepared for the following epitopes: g209-pa; g209-2M and Flu M1:58–66 (GILGFVFTL). All peptides were commercially synthesized and purified by gel filtration (Princeton Biomolecules, Columbus, OH). The refolding reaction was dialyzed and concentrated for purification of correctly refolded sHLA on gel filtration. Monomeric sHLA was biotinylated with BirA (Avidity, Denver, CO) at the heavy chain and separated from free biotin by gel filtration. Biotinylated sHLA was tetramerized by adding avidin-PE (Pierce, Rockford, IL) at a 4:1 molar ratio. The final concentration of tetramer was adjusted to 2 µg/ml for g209 and g209-2M tHLA and to 1 µg/ml for Flu tHLA. As examined by gel filtration, all tHLA were without detectable free avidin-PE. After overnight depletion of monocytes, nonadherent PBMC were resuspended at 106 cells/50 µl ice cold FACS buffer (phosphate buffer plus 5% inactivated FCS, Biofluids), and cells from day 10 CTL cultures were washed and resuspended at 2 x 105 cells/50 µl cold FACS buffer. Cells were incubated on ice with 1 µg tHLA for 15 min, and incubation was then continued for 30 min with 10 µl anti-CD8 mAb (Becton Dickinson, San Jose, CA). Cells were washed twice in 2 ml cold FACS buffer before analysis by FACS (Becton Dickinson). Two hundred thousand events were acquired. tHLA staining specificity was previously established by extensive analysis of T cell clones specific for each of the described epitopes and by comparative analysis of short term CTL cultures also specific for the above epitopes (11).
In vitro sensitization assessment of peptide-specific CTL reactivity
As previously described (5), cryopreserved PBMC
were thawed into CM. Cells were plated at 3 x
106 PBMC in 2 ml medium with 1 µM peptide. IL-2
(300 IU/ml) was added on day 2, and cells were harvested between days
11 and 13 after initiation of the culture. The harvested cells were
then stimulated withT2 cells pulsed with 1 µM peptide for 18–24 h at
37°C. IFN-
release into the supernatant was measured by a standard
ELISA assay. Reactivity was scored as positive (+) if IFN- release
was twice background and >100 pg/ml.
RNA isolation and cDNA synthesis
RNA isolation from PBMCs or fine needle aspirate biopsies was
performed in batches containing patient pre- and posttherapy samples
with RNeasy mini kits (Qiagen). The RNA was eluted with water and
stored at -70°C. For cDNA synthesis, 
1 µg total RNA was
transcribed with cDNA transcription reagents (Perkin-Elmer, Foster
City, CA) with the use of random hexamers. cDNA was stored at -30°C
until quantitative RT-PCR was performed.
Real time quantitative RT-PCR
Measurement of gene expression was performed utilizing the ABI
prism 7700 Sequence Detection System (Perkin-Elmer) as previously
described (12, 13). Primers and TaqMan probes (Custom
Oligonucleotide Factory, Foster City, CA) were designed to span
exon-intron junctions to prevent amplification of genomic DNA and to
result in amplicons <150 bp to enhance efficiency of PCR
amplification. TaqMan probes were labeled at the 5'-end with the
reporter dye molecule FAM (6-carboxyfluorescein; emission
max = 518 nm) and at the 3'-end with the
quencher dye molecule TAMARA (6-carboxytetramethylrhodamine; emission
max = 582 nm). cDNA standards were generated
by reverse transcriptase, primer-specific amplification of mRNA of the
relevant genes by a technique identical with the one used for the
preparation of test cDNA. Amplified cDNA was then purified and
quantitated by spectrophotometry (OD260). Copies
were calculated using the m.w. of each individual gene amplicon. RT-PCR
reactions of cDNA specimens and cDNA standards were conducted in a
total volume of 25 µl with 1x Taq Man Master Mix
(Perkin-Elmer) and primers and probes at optimized concentrations
(Table I
). Thermal cycler parameters included 2 min at 50°C, 10 min
at 95°C, and 40 cycles involving denaturation at 95°C for 15
s, annealing/extension at 60°C for 1 min. Real time monitoring of
fluorescent emission from cleavage of sequence specific probes by the
nuclease activity of taq polymerase allowed definition of
the threshold cycle during the exponential phase of amplification
(12). Standard curves were generated for each gene
quantitated and were found to have excellent PCR amplification
efficiency (90–100%; 100% indicates that in each cycle the amount of
template is doubled) as determined by the slope of the standard curves.
Linear regression analysis of all standard curves were 
0.99. Standard
curve extrapolation of copy number was performed for the gene of
interest as well as an endogenous reference gene for each sample.
Normalization of samples was performed by dividing the copies of the
gene of interest by copies of the reference gene. All PCR assays were
performed in duplicates and reported as the average. A 2-fold
difference in gene expression was found to be within the discrimination
ability of the assay (data not shown).
Direct molecular assessment of peptide and melanoma-specific CTL reactivity
Cryopreserved PBMC were thawed into CM as described
(5). To determine the optimal conditions for assessing
direct PBMC reactivity to the immunizing peptides, experiments were
conducted using PBMC obtained from patients after immunization. Peptide
concentrations ranging from 0.01 to 10 µM were evaluated, as were
harvest times ranging from 2 to 24 h, and optimal recovery time of
physiological cell metabolism for thawed PBMC. On the basis of these
optimization experiments (data not shown), direct PBMC assays were
conducted using 3 x 106 PBMC in 2 ml of
media, which were allowed to recover by incubation at 37°C in 5%
CO2 for 10 h. Either 1 µM peptide or
1 x 106 melanoma cells were then added to
the PBMC and incubated at 37°C in 5% CO2 for
2 h. No exogenous cytokines or other stimulants were added. The
cells were then harvested, and RNA isolation and cDNA transcription
were performed. Quantitative RT-PCR was performed for IFN- mRNA
expression and normalized to copies of CD8 mRNA from the same
sample.
Direct molecular assessment of gene expression within the tumor microenvironment
Sequential FNA of the same metastatic tumor site were performed before and after therapy. Aspirate material was examined and verified by a cytopathologist to contain melanoma cells. The remainder of the aspirate was placed in RNA lysis buffer (Qiagen) and stored at -180°C. RNA isolation and cDNA transcriptions from the aspirated material were performed in batches containing patient pre- and posttherapy samples to minimize variability. Quantitative RT-PCR was performed to assess changes in gene expression within the sequential tumor biopsies. Because the aspirates represent only a portion of any tumor and because the immune infiltrate can vary between individual parts of tumors, apparent changes in gene expression between pre- and postvaccination FNA aspirates could only reflect that different areas of the tumor had been probed over time. Thus, the data presented should be interpreted with caution until a larger patient population can be collected and analyzed.
Direct immunofluorescence of fine needle aspirate biopsies
Sequential fine needle aspirates obtained from the same metastatic tumor site were performed before and throughout therapy. Aspirated material was examined and verified by a cytopathologist to contain melanoma cells. Cytospins were performed, and the slides were fixed with acetone for 10 min. tHLA (1 µg) was added to specimens for 2 h at room temperature. Slides were washed vigorously with isotonic saline and visualized with fluorescent microscopy (Olympus, New Hyde Park, NY) at 576 nm.
Statistical analysis
Result reproducibility was tested performing a set of
consecutive experiments in which pre- and postvaccination PBMC obtained
from the same plasma pheresis from the same patient were independently
thawed, stimulated, and processed for cDNA preparation and qRT-PCR.
This set of experiments demonstrated that measurements of cytokine mRNA
expression were highly reproducible (Table II). Whereas no significant difference
could be noted among pre- and postvaccination PBMC that had not
received stimulation, a highly significant difference was noted after
stimulation of PBMC with 209-pa peptide (unpaired t test,
p < 0.001). Comparison of pre- vs posttreatment CTL
reactivity was assessed by a paired comparison of the fold increment in
IFN- transcript detection in response to stimulation of a given
sample over IFN- transcript detection in the same sample that had
not been stimulated. To address the accuracy of this method, 24 PBMC
samples obtained from HLA-A*0201-expressing patients who had never
received vaccination with 209-2M or 209-pa were tested. Furthermore, no
evidence of reactivity against either epitope could be detected by in
vitro sensitization assays in these samples. The ratio of IFN- mRNA
transcript detectable in epitope-stimulated PBMC to IFN- mRNA
transcript detectable in nonstimulated PBMC ranged from 0.6 to1.4 with
a median of 0.9 and a mean value of 0.94 ± 0.05 (SEM) at a 95%
confidence level of 0.09. To minimize the possibility of falsely
considering PBMC immunoreactive, we accepted a 2-fold increase in
stimulated-unstimulated IFN- transcript ratio (2.0 corresponding
to >5 SDs above the median) as evidence of epitope-specific
reactivity.
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transcript comparing pre- and postvaccination
samples were evaluated parametrically by a paired t test.
Correlation between gp100 transcript expression in FNA and fold
increase in IFN- mRNA detection after vaccination was compared by
simple linear regression analysis. |
Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess direct Ag recognition and reactivity by CTL in
peripheral blood, PBMC obtained by leukapheresis from patients before
and after two cycles of 209-2M vaccination were directly exposed ex
vivo to the 209-pa and 209-2M peptides or melanoma tumor cells. No
prior in vitro sensitization or culturing of lymphocytes was performed,
nor were exogenous cytokines added to the cells. Because of the low
frequency of 209-pa-reactive CTL in bulk PBMC, changes in cytokine
release after peptide elicitation were typically below the sensitivity
of standard ELISA assays. Therefore, we used real time quantitative
RT-PCR to monitor the PBMC for highly specific and quantitative changes
in gene expression. As illustrated in Fig. 1a, PBMC obtained from a patient
after two immunizations with the 209-2M peptide demonstrated a
phenotypic increase of CD8+ cells stained with
the 209-2M HLA tetramer, as well as the 209-pa HLA tetramer (0.2 and
0.12% of the total PBMC population, respectively). The direct
functional analysis of the preimmunized PBMC revealed no detectable
reactivity after incubation with either the modified 209-2M or the
native 209-pa peptides (Fig. 1b). However, the postimmunized
PBMC, within a 2-h peptide exposure, demonstrated significant increases
in mRNA for the CD69 CTL activation marker, the IL-2
receptor
(CD25), and the cytokines IFN-, TNF-, GM-CSF, and IL-2 (Fig. 1b). Peptide exposure did not result in changes in the gene
expression for IL-1, IL-1ß, IL-4, IL-5, IL-8, IL-12, or IL-15
(data not shown). We consistently noted that postimmunized PBMC
demonstrated greater induction of gene expression after exposure to the
modified 209-2M peptide when compared with 209-pa peptide (Fig. 1b). Physiologically, the cytokine kinetics of IFN-,
GM-CSF, and IL-2 showed strikingly similar characteristics but the
quantitative expression of IFN- mRNA was severalfold higher than
that of the other two genes. The favorable signal-noise ratio for
IFN- mRNA expression made this the most suitable single gene to
follow as a highly sensitive and specific marker of immune reactivity
in our subsequent studies. Reproducible results further delineated that
a 2–3 h elicitation time period with 1 µM of peptide to be the
optimal parameters for IFN- mRNA induction (data not shown). To
account for potential variability in the number of
CD8+ cells in the samples and reverse
transcriptase efficiency during cDNA preparation, normalization of
IFN- transcripts was performed by dividing by CD8 mRNA copies. CD8
mRNA expression was stable during experiments when compared with
traditional housekeeping genes such as ß-actin, GAPDH, and rRNA (data
not shown). To define the approximate sensitivity limit of this
molecular assay, in vitro cultured, 209-pa-reactive CTL clones were
spiked into nonreactive autologous PBMC. Significant 209-pa reactivity
(compared with response to an irrelevant melanoma Ag epitope, MART
27–35) could be seen at a spiked dilution of 1 CTL clone in 50,000
PBMC (Fig. 1c). To determine whether the observed peptide
reactivity was associated with tumor reactivity, bulk PBMC were exposed
directly to a panel of melanoma cell lines. Response from postimmunized
PBMC was found against two HLA-A2+/gp100+
melanomas (624.38 Mel and 888 Mel (A2+)), but not
against two HLA-A2-/gp100+ melanomas (624.28 Mel
and 888 Mel) as seen in Fig. 1d. Our cumulative observations
of peripheral lymphocytes demonstrated that 209-2M peptide
immunization could result in a significant increase in circulating
CTL with highly specific activity directed against a tumor Ag target.
Further, these findings were evident in cells, which were obtained
directly from patients without any prior in vitro manipulation.
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We next examined the relationship between the level of direct
209-pa peptide reactivity in peripheral blood and clinical tumor
response. Pre- and postvaccination PBMC were obtained from 10
consecutive patients who were vaccinated with 4 melanoma-associated
peptides that included 209-2M (see Materials and Methods).
PBMC were analyzed for 209-pa reactivity by direct molecular assay and
by standard cytokine release assays performed on in vitro sensitized
cultures (Table III). In these
experiments, evidence of 209-pa immunization for both assays was
defined as 209-pa epitope reactivity in postimmunized PBMC that was
measured to be twice the background (i.e., no peptide elicitation) and
not demonstrable in the preimmunized samples. Reactivity measured by
the IVS assay was qualitatively scored as positive (+) or negative (-)
because of the inherent in vitro variability of culture growth that
could influence quantitative analysis. We found that the IVS assay
could generate PBMC cultures with specific antipeptide reactivity
(IFN- cytokine release) in 7 of 10 patients after immunization,
while the direct molecular assay demonstrated immune reactivity
(IFN- mRNA production) in only 5 of the 10 patients. Patients i and
j (Table III) demonstrated qualitative evidence of culture reactivity
by the IVS assay but had less than a 2-fold increase by direct
molecular assay. Further, for the patients who demonstrated evidence of
immunization by both assays (patients a–e), direct analysis
found significant quantitative heterogeneity among the reactivities,
ranging from 2.1- to 76.7-fold over background. Thus, we observed that
patients who received identical vaccine therapies mounted very
different degrees of peripheral CTL response against the targeted Ag.
Despite these findings, there was no correlation between any level of
peripheral 209-pa reactivity and objective local tumor response with
immunization.
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To determine whether lack of tumor regression was due to lack of
localized immune response within the tumor, we examined 209-2M
therapy-related changes within the local tumor microenvironment of
subcutaneous melanoma metastases. Sequential fine needle aspirates
of individual lesions in situ were performed on 27 patients before and
after vaccine-based immunotherapy. These patients were divided into
3 cohorts based on their treatments (see Materials and
Methods): a 209-2M cohort (9 patients/11 lesions); and 2 control
groups, a non-209-2M cohort (10 patients/11 lesions) and an IL-2 cohort
(8 patients/9 lesions). The control therapies represented a variety of
experimental vaccine protocols that had shown no evidence of
immunization by standard in vitro sensitization assays of peripheral
blood. Quantitative RT-PCR was performed from RNA isolated directly
from aspirated material obtained from the same lesion before and after
treatment. No in vitro culturing or stimulation were performed on the
aspirate, so that a true representation of in vivo gene expression
and immune reactivity could be ascertained. As demonstrated in Fig. 2a, sequential biopsies from the
209-2M cohort showed statistically significant changes in 8 of 11
lesions having a 2-fold increase in IFN- mRNA expression
(normalized to CD8) when pretherapy levels were compared with
posttherapy levels for the same lesion (paired t test;
p = 0.01). In contrast, there was no significant change
in IFN- mRNA expression for the non-209-2M cohort (2 of 11 lesions,
p = 0.40) or the IL-2 cohort (3 of 9 lesions,
p = 0.19). Interestingly, direct quantitation of CD8
and CD4 mRNA (normalized to ß-actin mRNA), as a representation of
cellular immune infiltration in the biopsies, showed no significant
change in any of the cohorts (Fig. 2, b and c).
However, there was a slight trend toward significance for an increase
in CD8 mRNA in the IL-2 cohort (p = 0.06).
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mRNA expression, the magnitude of this increase was quite
variable among the individual lesions, with one lesion showing a
35-fold increase, whereas others showed marginal or no change. In an
effort to explain this heterogeneity, we examined the concurrent mRNA
expression for the targeted tumor Ag, gp100, in each of the biopsies.
As demonstrated in Fig. 2d, quantitative RT-PCR for gp100
mRNA expression (normalized to ß-actin mRNA) also demonstrated marked
heterogeneity, which correlated very highly with the observed tumor
changes in IFN- mRNA for the 209-2M cohort (r = 0.94,
p < 0.0001). This correlation was not seen in the
other two treatment cohorts, whose lesions were obtained and processed
in an identical manner and which showed similar variability of gp100 Ag
expression (Fig. 2, e and f). These findings
demonstrated that 209-2M immunization (in contrast to other therapies)
could result in measurable immunological changes within tumors if
appropriate levels of Ag were present. Change in IFN- mRNA was used
as a parameter rather than the absolute levels of IFN- transcript to
better represent the impact of therapy and to account for in vivo
baseline variability among lesions. Direct immunofluorescence of fine needle aspirate biopsies demonstrates localization of 209-pa reactive CTL
To visualize the cellular response within the tumor
microenvironment after 2092-M vaccination, we examined cytospins
prepared from sequential fine needle aspirates of the lesion which
showed the greatest change in IFN- mRNA levels. As shown in Fig. 3c, Wright-Giemsa staining of the
postimmunized sample demonstrated an abundance of intact large
nucleated melanoma tumor cells (pink staining). Concurrent staining
with the 209-pa HLA tetramer and examination with fluorescent
microscopy illustrated the phenotypic presence of g209-pa specific
lymphocytes within this lesion (Fig. 3d). In contrast, no
209-pa-specific T cells could be identified in the preimmunized
specimen (not shown).
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We next addressed whether peripheral and localized immune response
measured in the 209-2M cohort was sufficient to cause tumor regression.
Table IV documents individual lesion
sizes during 209-2M-based therapy and summarizes the simultaneous
analysis of peripheral blood and local tumor reactivity. PBMC
reactivity was found in 4 of 9 patients by the IVS assay and in 3 of 9
patients by the direct molecular assay. The magnitude of the direct
PBMC reactivity in these patients was low, ranging from 2.2- to
8.1-fold over background. As described in Fig. 2, direct tumor analysis
found evidence of immunization in 8 of the 11 lesions. Increases in
IFN- mRNA levels were apparent in 5 of these lesions (patients 2, 3,
4, 5, and 8) in the absence of measured peripheral blood reactivity by
direct molecular assay. Despite even these observed localized
responses, none of the 11 lesions showed significant regression in size
after vaccination.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, GM-CSF,
TNF-, and IL-2), and the proliferation marker CD25 was found
directly in postimmunized PBMC after immediate peptide exposure ex
vivo. This demonstrated that 209-2M vaccination could result in
circulating and functional CTL with activity against the targeted tumor
Ag. It is unclear why there was marked quantitative heterogeneity in
peripheral blood reactivity among patients receiving identical
immunization protocols with the 209-2M peptide. This finding may
reflect a biological variable among patients that could play an
important role in the optimization of future clinical vaccine
regimens.
Our analysis of sequential tumor biopsies during 209-2M-based
vaccination (either 209-2M or ES-209-2M; refer to Materials and Methods
for details) found that 8 of 11 lesions (73%) demonstrated significant
increases in IFN- mRNA (paired t test, p
= 0.01), whereas the control cohorts demonstrated no significant
change. Because 209-2M peptide immunization is highly specific in its
ability to generate an epitope-specific CTL response, we believe that
the changes in the biopsy IFN- mRNA levels are indicative of
enhanced specific lymphocyte reactivity within the tumor
microenvironment. Further, because the tumor-derived IFN- mRNA
levels were measured without ex vivo stimulation, we believe that these
cytokine mRNA levels are representative of the natural in vivo
interaction between vaccine-induced CTL and endogenous tumor Ag. This
conclusion is supported by the strong correlation between IFN- and
gp100 mRNA expression observed in the 209-2M cohort (r
= 0.94, p < 0.0001). Interestingly, five tumor lesions
showed treatment-related changes in the absence of detectable
peripheral blood reactivity. Local immune changes without detectable
systemic precursor reactivity may imply a greater Ag-specific CTL
frequency in the local tumor microenvironment than in bulk PBMC, as
suggested previously (15). Because of these findings, we
believe that sequential analysis of tumors represents the most
sensitive and relevant approach to analyzing in vivo effects of cancer
vaccines. Further, this molecular methodology would be ideally suited
for the assessment of a variety of novel biological agents in clinical
trials, if appropriate target tissue is easily accessible.
Unfortunately, despite our evidence of immune reactivity in peripheral
blood and at the local tumor site, there was no significant impact of
209-2M therapy on tumor viability and progression. Given that the
increase in IFN- mRNA in tumors was noted in the absence of
significant CD8 and CD4 mRNA increases, we hypothesize that a
classically induced response of cellular recruitment and inflammation
likely did not occur in these lesions. These results demonstrate the
presence but perhaps the limited effectiveness of vaccine-induced T
cell response within the target environment. It is possible that the
characteristics of Ag presentation exercised by tumors are not optimal
to maintain T cells in a state of activation at the tumor site. It has
been suggested that tumors induce tolerance by presenting
epitope-specific stimulation (signal 1) without costimulation (signal
2) to wandering memory T cells (16). Of interest, our
initial evaluation has shown minimal evidence of IL-2 mRNA, a critical
growth factor for T cell proliferation, in the tumor microenvironment
of vaccine-treated patients (work in progress). Some models predict
that in the absence of an ongoing "danger signal," the vaccination
response will wane and eventually stop (17). Tumor escape
through Ag and/or HLA loss (18) does not fully explain our
observed tumor resistance to therapy, given that we had lesions with
documented high expression of gp100 and HLA A2 showing no preferential
regression. Other possible explanations include the effects of local
immunosuppressive factors such as IL-10 and TGF-ß (19)
and expression of apoptotic signals (20). Recently, it has
been reported that effector T cells induced against antigenic tumors
could be maintained by prolonged or repetitive vaccination
(21). Modifications and adjuvants to immunization schemes,
such as exogenous IL-2 and modalities to facilitate CTL help, may
provide additional stimuli to heighten the immune reactivity that we
have observed into effective antitumor response. Direct serial
molecular analysis of gene expression in tumors represents an extremely
sensitive and powerful tool to monitor these immunological changes in
vivo. This methodology may help further guide the development of future
biological therapies.
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Footnotes |
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2 Abbreviations used in this paper: g209-2 M, gp100:209217 (210 M); IVS, in vitro sensitization; pa, parental; FNA, fine needle aspiration; CM, culture medium; sHLA, soluble HLA.
Received for publication July 20, 1999. Accepted for publication September 24, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 R. E. Nair, M. O. Kilinc, S. A. Jones, and N. K. Egilmez Chronic Immune Therapy Induces a Progressive Increase in Intratumoral T Suppressor Activity and a Concurrent Loss of Tumor-Specific CD8+ T Effectors in her-2/neu Transgenic Mice Bearing Advanced Spontaneous Tumors. J. Immunol., June 15, 2006; 176(12): 7325 - 7334. [Abstract] [Full Text] [PDF]
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K. Rezvani, J. M. Brenchley, D. A. Price, Y. Kilical, E. Gostick, A. K. Sewell, J. Li, S. Mielke, D. C. Douek, and A. J. Barrett T-Cell Responses Directed against Multiple HLA-A*0201-Restricted Epitopes Derived from Wilms' Tumor 1 Protein in Patients with Leukemia and Healthy Donors: Identification, Quantification, and Characterization Clin. Cancer Res., December 15, 2005; 11(24): 8799 - 8807. [Abstract] [Full Text] [PDF]
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C. J. Wheeler, A. Das, G. Liu, J. S. Yu, and K. L. Black Clinical Responsiveness of Glioblastoma Multiforme to Chemotherapy after Vaccination Clin. Cancer Res., August 15, 2004; 10(16): 5316 - 5326. [Abstract] [Full Text] [PDF]
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J. S. Yu, G. Liu, H. Ying, W. H. Yong, K. L. Black, and C. J. Wheeler Vaccination with Tumor Lysate-Pulsed Dendritic Cells Elicits Antigen-Specific, Cytotoxic T-Cells in Patients with Malignant Glioma Cancer Res., July 15, 2004; 64(14): 4973 - 4979. [Abstract] [Full Text] [PDF]
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M. Grube, K. Rezvani, A. Wiestner, H. Fujiwara, G. Sconocchia, J. J. Melenhorst, N. Hensel, G. E. Marti, L. W. Kwak, W. Wilson, et al. Autoreactive, Cytotoxic T Lymphocytes Specific for Peptides Derived from Normal B-Cell Differentiation Antigens in Healthy Individuals and Patients with B-Cell Malignancies Clin. Cancer Res., February 1, 2004; 10(3): 1047 - 1056. [Abstract] [Full Text] [PDF]
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C. J. Wheeler, K. L. Black, G. Liu, H. Ying, J. S. Yu, W. Zhang, and P. K. Lee Thymic CD8+ T Cell Production Strongly Influences Tumor Antigen Recognition and Age-Dependent Glioma Mortality J. Immunol., November 1, 2003; 171(9): 4927 - 4933. [Abstract] [Full Text] [PDF]
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 K. Rezvani, M. Grube, J. M. Brenchley, G. Sconocchia, H. Fujiwara, D. A. Price, E. Gostick, K. Yamada, J. Melenhorst, R. Childs, et al. Functional leukemia-associated antigen-specific memory CD8+ T cells exist in healthy individuals and in patients with chronic myelogenous leukemia before and after stem cell transplantation Blood, October 15, 2003; 102(8): 2892 - 2900. [Abstract] [Full Text] [PDF]
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C. Rentzsch, S. Kayser, S. Stumm, I. Watermann, S. Walter, S. Stevanovic, D. Wallwiener, and B. Guckel Evaluation of Pre-existent Immunity in Patients with Primary Breast Cancer: Molecular and Cellular Assays to Quantify Antigen-Specific T Lymphocytes in Peripheral Blood Mononuclear Cells Clin. Cancer Res., October 1, 2003; 9(12): 4376 - 4386. [Abstract] [Full Text] [PDF]
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 G. Q. Phan, J. C. Yang, R. M. Sherry, P. Hwu, S. L. Topalian, D. J. Schwartzentruber, N. P. Restifo, L. R. Haworth, C. A. Seipp, L. J. Freezer, et al. Cancer regression and autoimmunity induced by cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma PNAS, July 8, 2003; 100(14): 8372 - 8377. [Abstract] [Full Text] [PDF]
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G. J. Ullenhag, J.-E. Frodin, S. Mosolits, S. Kiaii, M. Hassan, M. C. Bonnet, P. Moingeon, H. Mellstedt, and H. Rabbani Immunization of Colorectal Carcinoma Patients with a Recombinant Canarypox Virus Expressing the Tumor Antigen Ep-CAM/KSA (ALVAC-KSA) and Granulocyte Macrophage Colony- stimulating Factor Induced a Tumor-specific Cellular Immune Response Clin. Cancer Res., July 1, 2003; 9(7): 2447 - 2456. [Abstract] [Full Text] [PDF]
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C. Palena, J. Schlom, and K.-Y. Tsang Differential Gene Expression Profiles in a Human T-cell Line Stimulated with a Tumor-associated Self-peptide versus an Enhancer Agonist Peptide Clin. Cancer Res., May 1, 2003; 9(5): 1616 - 1627. [Abstract] [Full Text] [PDF]
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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]
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T. Weinschenk, C. Gouttefangeas, M. Schirle, F. Obermayr, S. Walter, O. Schoor, R. Kurek, W. Loeser, K.-H. Bichler, D. Wernet, et al. Integrated Functional Genomics Approach for the Design of Patient-individual Antitumor Vaccines Cancer Res., October 15, 2002; 62(20): 5818 - 5827. [Abstract] [Full Text] [PDF]
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 G. Parmiani, C. Castelli, P. Dalerba, R. Mortarini, L. Rivoltini, F. M. Marincola, and A. Anichini Cancer Immunotherapy With Peptide-Based Vaccines: What Have We Achieved? Where Are We Going? J Natl Cancer Inst, June 5, 2002; 94(11): 805 - 818. [Abstract] [Full Text] [PDF]
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V. Monsurro, D. Nagorsen, E. Wang, M. Provenzano, M. E. Dudley, S. A. Rosenberg, and F. M. Marincola Functional Heterogeneity of Vaccine-Induced CD8+ T Cells J. Immunol., June 1, 2002; 168(11): 5933 - 5942. [Abstract] [Full Text] [PDF]
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 I. C. Le Poole, A. I. Riker, M. E. Quevedo, L. S. Stennett, E. Wang, F. M. Marincola, W. M. Kast, J. K. Robinson, and B. J. Nickoloff Interferon-{gamma} Reduces Melanosomal Antigen Expression and Recognition of Melanoma Cells by Cytotoxic T Cells Am. J. Pathol., February 1, 2002; 160(2): 521 - 528. [Abstract] [Full Text] [PDF]
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H. T. Khong and S. A. Rosenberg Pre-Existing Immunity to Tyrosinase-Related Protein (TRP)-2, a New TRP-2 Isoform, and the NY-ESO-1 Melanoma Antigen in a Patient with a Dramatic Response to Immunotherapy J. Immunol., January 15, 2002; 168(2): 951 - 956. [Abstract] [Full Text] [PDF]
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A. Perez-Diez, P. J. Spiess, N. P. Restifo, P. Matzinger, and F. M. Marincola Intensity of the Vaccine-Elicited Immune Response Determines Tumor Clearance J. Immunol., January 1, 2002; 168(1): 338 - 347. [Abstract] [Full Text] [PDF]
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G. A. Ohnmacht, E. Wang, S. Mocellin, A. Abati, A. Filie, P. Fetsch, A. I. Riker, U. S. Kammula, S. A. Rosenberg, and F. M. Marincola Short-Term Kinetics of Tumor Antigen Expression in Response to Vaccination J. Immunol., August 1, 2001; 167(3): 1809 - 1820. [Abstract] [Full Text] [PDF]
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K. Schumacher, W. Haensch, C. Röefzaad, and P. M. Schlag Prognostic Significance of Activated CD8+ T Cell Infiltrations within Esophageal Carcinomas Cancer Res., May 1, 2001; 61(10): 3932 - 3936. [Abstract] [Full Text]
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T. M. Clay, A. C. Hobeika, P. J. Mosca, H. K. Lyerly, and M. A. Morse Assays for Monitoring Cellular Immune Responses to Active Immunotherapy of Cancer Clin. Cancer Res., May 1, 2001; 7(5): 1127 - 1135. [Abstract] [Full Text]
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V. Monsurro, M.-B. Nielsen, A. Perez-Diez, M. E. Dudley, E. Wang, S. A. Rosenberg, and F. M. Marincola Kinetics of TCR Use in Response to Repeated Epitope-Specific Immunization J. Immunol., May 1, 2001; 166(9): 5817 - 5825. [Abstract] [Full Text] [PDF]
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K. Shimizu, E. K. Thomas, M. Giedlin, and J. J. Mulé Enhancement of Tumor Lysate- and Peptide-pulsed Dendritic Cell-based Vaccines by the Addition of Foreign Helper Protein Cancer Res., March 1, 2001; 61(6): 2618 - 2624. [Abstract] [Full Text]
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 T. S. Weiser, G. A. Ohnmacht, Z. S. Guo, M. R. Fischette, G. A. Chen, J. A. Hong, D. M. Nguyen, and D. S. Schrump Induction of MAGE-3 expression in lung and esophageal cancer cells Ann. Thorac. Surg., January 1, 2001; 71(1): 295 - 302. [Abstract] [Full Text] [PDF]
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M. V. Dhodapkar, J. W. Young, P. B. Chapman, W. I. Cox, J. F. Fonteneau, S. Amigorena, A. N. Houghton, R. M. Steinman, and N. Bhardwaj Paucity of Functional T-Cell Memory to Melanoma Antigens in Healthy Donors and Melanoma Patients Clin. Cancer Res., December 1, 2000; 6(12): 4831 - 4838. [Abstract] [Full Text]
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U. S. Kammula, F. M. Marincola, and S. A. Rosenberg Real-Time Quantitative Polymerase Chain Reaction Assessment of Immune Reactivity in Melanoma Patients After Tumor Peptide Vaccination J Natl Cancer Inst, August 16, 2000; 92(16): 1336 - 1344. [Abstract] [Full Text] [PDF]
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M.-B. Nielsen, V. Monsurro, S. A. Migueles, E. Wang, A. Perez-Diez, K.-H. Lee, U. Kammula, S. A. Rosenberg, and F. M. Marincola Status of Activation of Circulating Vaccine-Elicited CD8+ T Cells J. Immunol., August 15, 2000; 165(4): 2287 - 2296. [Abstract] [Full Text] [PDF]
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M. C. Panelli, M. P. Bettinotti, K. Lally, G. A. Ohnmacht, Y. Li, P. Robbins, A. Riker, S. A. Rosenberg, and F. M. Marincola A Tumor-Infiltrating Lymphocyte from a Melanoma Metastasis with Decreased Expression of Melanoma Differentiation Antigens Recognizes MAGE-12 J. Immunol., April 15, 2000; 164(8): 4382 - 4392. [Abstract] [Full Text] [PDF]
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), + 209-2M (
), postimmunized PBMC (
) +
209-pa (
) + 209-2M (
). c, Sensitivity of direct
molecular assay; 209-pa-reactive T cell clone was spiked into
preimmunized PBMC. Elicitation of 209-pa reactivity (IFN-
