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*
Surgery Branch, National Cancer Institute,
Laboratory of Cellular and Molecular Immunology, National Institute of Allergy and Infectious Diseases, and
Department of Transfusion Medicine, Clinical Center, 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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-galactosidase
(-gal)-expressing tumor cells, C25.F6, vaccinated them with
-gal-carrying viral vectors, and used quantitative RT-PCR to measure
the vaccine-induced immune response of splenocytes directly ex vivo. We
found that the strength of the response increased with increasing doses
of -gal-carrying vector and/or upon boosting with a heterologous
-gal-carrying virus. Most importantly, we found that the strength of
the detected immune response against this foreign Ag strongly
correlated with reduction in the number of lung metastases. The results
from this mouse model have major implications for the implementation of
tumor vaccines in humans. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thus, to determine, in principle, whether tumor rejection depends on
the strength of a vaccine-induced response as opposed to its mere
presence, we developed a mouse model in which the recipients are
immunologically functional and where other factors can be standardized
by homogeneity of the reagents and procedures adopted. In this
simplified model, we assessed whether the strength of the immune
response to an Ag expressed by tumor cells could be used as a marker to
predict the clinical outcome. By implanting known numbers of a stable
tumor clone into a set of inbred animals, heterogeneity due to the
genetic instability of neoplasia, or to the variability of the host,
was minimized. We chose a -galactosidase
(-gal)2-transduced
tumor, in which two heterologous fowl pox-based viral
vectors carrying the -gal Ag were able, in previous studies, to cure
lung metastases in 80% of mice (20). In this model,
specific CD8+ T cells that recognize an
H-2Ld restricted epitope (TPHPARIGL) appear
essential for tumor rejection. We then developed various sets of
primers and probes suitable for real-time quantitative RT-PCR
(qRT-PCR)-based direct quantification of -gal-specific
CD8+ T responses in splenocytes of vaccinated
mice. Using this model, we observed that the strength of the response,
as measured directly ex vivo, does indeed correlate with tumor
rejection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice, 6–10 wk old, were obtained from the Animal Production Colonies, National Cancer Institute-Frederick Cancer Research and Development Center, National Institutes of Health (Frederick, MD). The mice received care in accordance with the guidelines set forth by the Animal Research Advisory Committee of the National Institutes of Health. The National Institutes of Health is an American Association for the Accreditation of Laboratory Animal Care-accredited institution.
Tumor cell lines
CT26 is a colon carcinoma of BALB/c origin. CT26.CL25 is a
-gal-expressing cell line previously described (21).
C25.F6 (F6) is a stable clone expressing high levels of -gal
obtained as follows. High expressing -gal CT26.CL25 cells were
sorted and cultured at 1 cell/well without G418. After a first
screening, we again sorted the highest expressing -gal cells from
the positive clones and cultured them in the absence of G418 to assess
genetic stability. After a second screening, F6 was selected among
other clones because of its stable expression of high levels of
-gal. Cell lines were maintained in complete medium consisting of
RPMI 1640, 10% heat-inactivated FCS (Biofluids, Rockville, MD), 0.03%
L-glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin,
and 50 µg/ml gentamicin sulfate (National Institutes of Health Media
Center).
Detection and sorting of -gal-expressing cells by FACS
To detect and sort -gal-expressing tumor cells, we used the
fluorescein di--galactopyranoside (FDG) assay procedure
(22).
Peptides
The peptide TPHPARIGL, a -gal epitope presented by
H-2Ld Ags (aa 876–884), was synthesized by
Macromolecular Resources (Colorado State University, Fort Collins, CO)
to a purity of >95%. The influenza nucleoprotein peptide (QL9)
QLSPFPFDL, also presented by H-2Ld, was
synthesized using standard F-moc
(N-a-9-fluorenylmethoxycarbonyl) chemistry.
Recombinant viruses
The -gal-expressing recombinant vaccinia (VV) and
recombinant fowl pox (FPV) viruses have been previously described
(20). As control vaccination, we used the same recombinant
viruses, but lacking the LacZ gene (cVV and cFPV,
respectively).
In vivo treatment of lung nodules
BALB/c mice were challenged i.v. with 105
F6 tumor cells to establish lung nodules. Three days later, the mice
were primed with either no immunogen (untreated mice),
107 PFU of cFPV, or serial 10-fold dilutions of
FPV administered i.v. or i.p. as described in each experiment.
Fifteen days after tumor inoculation, each group was boosted with the
same amount of either no immunogen, cVV, or VV. Seven days after the
second vaccination, mice were sacrificed and metastatic lung nodules
were enumerated in a randomized manner. Splenocytes from randomly
chosen individual mice from each experimental group were analyzed for
ex vivo quantification of anti--gal-specific reactivity by
qRT-PCR.
Ex vivo quantification of anti--gal-specific
reactivity by real-time qRT-PCR
Splenocytes from untreated or vaccinated mice (5 x
105 cells) were plated in 100 µl of medium in
U-bottom 96-well plates and incubated overnight at 37°C in 5%
CO2. Peptide (1 µM) or tumor cells (1 x
105) were then added in a total volume of 200
µl/well and incubated at 37°C in 5% CO2 for
2 h. The cells were then harvested for RNA isolation with RNeasy
mini kits (Qiagen, Santa Clarita, CA) and cDNA transcription, with cDNA
transcription reagents (PerkinElmer, Foster City, CA) using random
hexamers. qRT-PCR was performed for IL-2, IL-4,
IL-10, TNF-
, and IFN-
mRNA
expression and normalized to copies of CD8 mRNA, e.g., the
number of copies of cytokine mRNA/the number of copies of CD8
mRNA.
Real-time qRT-PCR
Quantification of gene expression was performed using the ABI
prism 7700 sequence detection system (PerkinElmer) as previously
described (12). TaqMan probes were labeled at the 5' end
with the reporter dye molecule 6-carboxyfluorescein and at the 3' end
with the quencher dye molecule 6-carboxytetramethylrhodamine. RT-PCR of
cDNA test specimens and cDNA standards were conducted in a total volume
of 25 µl with 2x TaqMan master mix (PerkinElmer) and primers and
probes at optimized concentrations (200 and 100 nM, respectively).
Real-time monitoring of fluorescence emission from cleavage of
sequence-specific probes by Taq polymerase allowed
definition of the cycle threshold (Ct, the cycle at which fluorescence
exceeds 10 times the SD above the mean of the background) during the
exponential phase of amplification (23). Standard curves
were generated for each gene quantified and the reagents were found to
have excellent PCR amplification efficiency as determined by the slope
of the standard curves. Linear regression analysis showed that all
standard curves had R 
0.99. Standard curve extrapolation
of a copy number was performed for the gene of interest as well as for
the endogenous reference gene (CD8) for each sample.
Normalization of samples was performed by dividing the copies of the
gene of interest by the copies of the CD8 reference gene.
All PCR assays were performed in duplicate and their Ct values (SD
< 0.5) are reported as the average.
Statistical analysis
The mean and the SD of the number of lung metastases and the
ratio of IFN- for each group of mice were calculated. Statistical
significance of the observed differences was assessed by two-tailed
Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To develop a method for the quantitative ex vivo measurement of
-gal-specific responses generated in mice, we designed primers and
probes for mouse IL-2, IL-4, IL-10,
TNF-, IFN-, and CD8 genes (Fig. 1
a) to be used in qRT-PCR.
Fig. 1b shows a sample of the plots for 10-fold dilutions of
the standard for TNF-. Samples containing high
concentrations of cDNA (large circles) reach the threshold cycle
earlier than samples with low amounts of cDNA (smaller circles). We
designated Ct, on the linear portion of the curve, as that cycle in
which the fluorescence reaches 10 SD above the mean of the general
background (Fig. 1b). A Ct of 40 means that there was no
fluorescence detected in that sample after 40 cycles of PCR, therefore
the gene of interest was not present in the sample or was too low to be
detected. The formula for the standard curves is Ct = m
x (log10 copies) + b, where
the slope (m) reflects the efficiency of replication for a
particular pair of primers and the Y-intercept (b) reflects
the sensitivity of detection for a particular probe. When we plotted
the Ct for each sample of our gene standards against the
log10 of the copy number, the curves had a slope
between -3.18 and -3.32 (Fig. 1c) approaching the maximal
theoretical efficiency of the PCR (perfect doubling at each cycle
= -3.33). This indicated that our chosen primers were nearly maximally
efficient. Further, the extrapolated Y-intercepts lay near 40 cycles,
indicating that the lower level of detection for each combination of
primers and probes was between one and one hundred copies of the
relevant gene.
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-gal-specific
immune reactivity in the absence of any expansion or in vitro culture
steps, we injected two mice i.v. with 107 PFU of
FPV and two mice with the same amount of non--gal-carrying cFPV,
boosted them 14 days later with 107 VV or cVV,
respectively, and harvested the splenocytes 7 days later. We rested
5 x 105 splenocytes/well overnight in
96-well plates in the absence of growth factors and stimulated the
splenocytes the following day for 2 h with the
H-2Ld-restricted -gal peptide or the
-gal-expressing tumor F6 cells. As controls, the splenocytes were
stimulated with a peptide from influenza (Flu) that is also presented
by H-2Ld Ags or with the -gal-negative
parental tumor cells, CT26. We calculated -gal-specific reactivity
as the ratio of IFN- mRNA to CD8 mRNA in -gal-stimulated wells vs
control (Flu or CT26)-stimulated wells.
Table I shows the cytokine
production pattern in splenocytes from one representative mouse of two
tested, after 2 h of in vitro sensitization with -gal or Flu
peptide. No -gal-specific IL-2, TNF-, or
IFN- mRNA production could be detected in mice vaccinated
with control viruses (ratios -gal/Flu were <2). However, a
significant amount of -gal-specific IFN- transcript
was detected in splenocytes of -gal-vaccinated mice. For example,
the number of copies of mRNA found in the -gal stimulated sample of
a vaccinated mouse (247,035), when normalized to the copies of mRNA
detected for CD8 in the same well (25,442), equals 9.7. This
number represents the IFN- production, with respect to the number of
CD8 cells, upon stimulation with the -gal CD8 epitope. When compared
with the IFN- production in splenocytes from the same mouse
stimulated with the control Flu CD8 epitope (5,705 copies of
IFN-:20,339 copies of CD8 = 0.28), it is clear this vaccinated
mouse makes a 35-fold stronger response to -gal than to Flu. The
ratio of -gal-Flu calculated for the second -gal vaccinated mouse
was 51. Production of IL-2 and TNF- in the -gal and Flu
stimulated samples were calculated in the same way using their own
standard curves (Fig. 1
c) to extrapolate the cytokine gene
copy number from the Ct. As previously seen in PBMCs from vaccinated
patients (12), IL-2 and TNF- mRNA were also specifically
produced by splenocytes of -gal vaccinated mice, although to a lower
degree than IFN- (ratios of 4.8 and 4.0 -gal specific-Flu
specific, respectively, for the first mouse, and ratios of 25 and 3.3,
respectively, for the second mouse). As expected for responses to FPV,
no specific IL-10 or IL-4 production was detectable.
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-gal sensitization in an 8-h in vitro kinetic study (data not
shown), suggesting that in activated CTL, the release of these
effectors molecules is most likely modulated at the posttranscriptional
level. Because of its response to vaccination and to short-term in
vitro stimulation, we selected IFN- as the most sensitive marker for
the detection of -gal-specific responses ex vivo.
The vaccine system: anti--gal reactivity rises in response
to gradual increases in vaccine dose and number
We next evaluated a spectrum of vaccine doses to determine whether
there is a range in which the response would vary proportionately with
the dose. We vaccinated seven groups of mice i.v. with different
amounts of FPV (three mice per group received
102 or 103 PFUs and five
mice per group received from
104–108 PFUs). Seven days
later, we tested the splenocytes from one mouse per group to quantify
specific -gal reactivity by qRT-PCR-based measurement of IFN-
expression after stimulation with -gal peptide or the F6 tumor, as
described above. To determine whether a second dose would influence the
response, we boosted two mice from each experimental group i.v. 14 days
after the first vaccination. As it is thought that the Ab response to a
viral vector may inhibit its efficacy (20), we changed to
a vaccinia recombinant, each boosted mouse receiving a dose of VV
equivalent to the dose of FPV with which it had been previously
vaccinated (from 102 to 108
PFUs). As controls, two mice from each experimental group that had
received 104 to 108 PFUs of
FPV were injected with corresponding PFUs of control cVV instead of
VV. Seven days after the last vaccination, we measured the specific
-gal response from the splenocytes of these mice.
We found that, after a single vaccination, the magnitude of the immune
response began to increase with incrementally increasing viral doses
above 105 PFUs and had not yet reached a plateau
at doses of 108 PFUs (Fig. 2a); this did not improve, and
may have been somewhat inhibited by, boosting with a control vaccinia
vector (Fig. 2b). In response to a second vaccination with
VV, groups given 105 or more PFUs reached
plateau levels of IFN- production that were 5–20 times higher than
mice given a single vaccination (Student’s t test,
p = 0.05 for PFU 5; Fig. 2c). Thus, using
direct ex vivo measurements, we found that doses between
103 and 107 PFUs of
-gal-carrying vectors represented a frequency and range of
vaccination that resulted in significant differences in
vaccine-specific immune responsiveness, as measured directly ex
vivo.
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To test whether the incremental changes in vaccine-induced
responsiveness, as measured by IFN- production ex vivo, have any
meaningful correlation with in vivo anti-tumor activity, we tested
our vaccine protocols on mice carrying a tumor that expresses -gal.
Previous studies with the -gal-expressing tumor CT26.CL25 have shown
that vaccination with -gal-expressing viral vectors can have some
effect on tumor growth. However, the effects were not complete, as 20%
of the tumors eventually grew out, despite vaccination
(20). After analyzing these studies, we considered the
possibility that the -gal-transfected cell line that had been used
might not have comprised a clone of stably transfected cells but a
mixed population from which nontransfected cells, or perhaps loss
variants, might have escaped from the vaccine-induced immune response.
This might have obscured the relationship between vaccination and tumor
rejection. In fact, when we looked at -gal expression in cell line
CT26.CL25 cultured in the presence (Fig. 3a) or absence (Fig. 3b) of selection medium, we detected a cell population that
completely lost -gal expression when line CT26.CL25 was cultured
without G418 (Fig. 3b). Therefore, we set out to determine
whether we could select for a stable -gal expressing clone of
CT26.CL25 with which to continue our vaccination study. We first
selected, by FACS sorting and single cell cloning, several cells among
the top 1% of the highest -gal expressors. We grew the resulting
clones without G418 selection medium to allow unstable clones to lose
expression, then we sorted again. Fig. 3, c and
d, demonstrates the levels of -gal expression by clone
CT26.CT25.F6 7 days and 2 mo after the second sorting when cultured in
the absence of selection medium. Although the level of -gal
expression dropped somewhat,the cells did not completely lose -gal
expression, even after prolonged in vitro culture without selective
agents, suggesting a decreased likelihood that it would lose -gal
expression during a similar period of in vivo growth. Therefore, we
chose clone F6 to continue our vaccination study.
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production, correlates with tumor rejection, we injected
incrementally increasing primary and boosting doses of -gal vectors,
as in Fig. 2c, into mice bearing 3-day-old pulmonary
metastases of clone F6. Seven days after the last vaccination, we
sacrificed the mice, counted the number of pulmonary metastases, and
measured the IFN- response against -gal from their splenocytes.
Results from two similar experiments (Fig. 4) show that there was a clear dose
response in effective tumor clearance, as the number of metastases
dropped with escalating doses of -gal-carrying vaccine vectors.
Moreover, this correlated with a concomitant increase in specific
IFN- production against -gal peptide and the F6 tumor that was
measured in randomly selected mice from each treatment group. Notably,
there was a significant amount of variability in specific IFN-
production among mice in the same treatment group and this variability
revealed that the strength of the measured ex vivo response correlated
more closely with in vivo tumor immunity than we might have expected
from a simple dose escalation trial. For example, in experiment one,
one mouse in the group vaccinated with 102 PFUs
(designated by 
) made no specific IFN- in response to -gal
(Fig. 4b; a ratio of 1.1) and had 35 metastases whereas
another in the same group (
) had a specific IFN- ratio of 2.8 and
no metastases. Such correlations suggest that different individuals
respond differently to a particular dose of vaccine and that ex vivo
IFN- production is a good representation of the in vivo response to
the tumor.
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), although i.p. injection was
10-fold less efficient than i.v. administration. As measured by
direct ex vivo IFN- mRNA expression, the response to -gal was
first detected in mice vaccinated with 104 PFUs.
By this route of vaccination, the ex vivo test again correlated very
well with tumor rejection, as those groups responding more strongly
also contained fewer tumor metastases.
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mRNA production. To better compare the antitumoral
effect among mice from different experiments, we normalized the number
of metastases carried by each mouse to the average of metastases in the
untreated control group from the same experiment. Fig. 6a shows the relationship
between tumor treatment and vaccine dose in mice vaccinated twice i.v.
(•) or i.p. () with -gal-carrying vectors, while Fig. 6b shows the relationship between tumor treatment and ex
vivo-specific IFN- production. This plot includes mice vaccinated
with different doses and numbers of -gal vaccines and using
different routes of administration (i.p., ; i.v., •). Overall, it
is clear that there is a strong correlation between the dose of
administered vaccine, the ex vivo IFN- response, and the reduction
in tumor metastases, whether the vaccine was given i.v. or i.p. There
are two noteworthy features apparent in this summary. The first is that
(perhaps not surprisingly), the route of vaccination had a strong
impact, both on ex vivo responsiveness, and on reduction of tumor load.
i.v. administration was 10 to 100-fold more effective than i.p. The
second is that individual animals reacted quite differently to the same
dose of vaccine, even though they were age-matched members of the same
inbred strain. This variation underscored the value of measuring the ex
vivo production of IFN-, as it showed that IFN- production
correlates more closely with tumor reduction than does vaccine dose,
number, or route of administration. For example, the mouse inside an
open diamond made an Ag-specific ratio of 2.8 IFN- units and
completely rejected the tumor, although it had only received the lowest
dose of 102 PFU of -gal-viruses. In contrast,
the mice inside open triangles showed poor anti-tumor effect (lung
metastases = 81 and 80% of controls), although one received
102 PFU of -gal viruses (small triangle) and
the other one received 10 times more (large triangle). However, their
specific IFN- responses were similarly low (ratio IFN- = 0.5
and 0.9, respectively). The correlation is equally striking in mice
vaccinated by the i.p. route. Although this form of vaccination was 10
to 100-fold less effective than i.v. administration, the correlation
with IFN- production was indistinguishable from that of mice that
had been vaccinated i.v. For example, though the two mice delineated by
circles were given 103 PFU i.v. (small circle) or
105 PFU i.p. (large circle), they made similar
ratios of 2.8 and 3.1 IFN- units and rejected their tumors. In the
middle ranges, though the two mice delineated by squares were given
102 PFU i.v. (small square) or
104 PFU i.p. (big square), they showed weak ex
vivo specific IFN- production of 1.4 and 1.7 units, respectively,
and weak anti-tumor effectiveness with 65 and 30% of lung
metastases, respectively. These data show that the immediate ex vivo
measurement of Ag-specific IFN- production is an excellent
representation of the effective in vivo anti-tumor response.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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did not seem to correlate with
the progression of tumors in vaccinated patients. In many cases, the
tumors persisted despite measurable vaccine-induced immunity while in
others, the tumors remained static or regressed in the absence of a
large IFN- response (12). Although the possibility
existed that there truly was no connection between our in vitro
measurement and the in vivo response, we hypothesized that other
confounding features (such as patient variability, tumor heterogeneity,
and the immunosuppressive effects of prior anti-tumor therapies)
might be obscuring the picture. To determine, in principle, whether the
strength of the in vivo response to a tumor vaccine can be measured by
an immediate ex vivo assay, whether this measurement has any relevance
to tumor clearance and whether the response can be manipulated by
varying the parameters of vaccination, we created a simple mouse model
in which several technical details allowed us to minimize the kind of
variability that is seen with human patients. First, we used
age-matched inbred (BALB/c) mice to minimize host variability. Second,
we used a cloned and recloned -gal-transfected tumor to minimize
tumor heterogeneity. Transfected cell lines are initially notoriously
unstable and, in some previous studies, the transfected tumors may have
been mixed populations at the time of injection (20, 24, 25, 26). Thus, tumor outgrowth might as easily have been due to
escape by cells that had never expressed the transfected gene as to
mutants that had lost expression. By selecting a stable transfectant
and recloning it several times, we minimized the outgrowth of
non-Ag-expressing tumors. Third, to minimize the potentially
suppressive effect of Ab to the viral vectors, we used different viral
vectors for the primary and secondary vaccinations. Fourth, we took
advantage of the sensitivity of real-time qRT-PCR to measure the ex
vivo response of vaccinated cells after only 2 h of stimulation in
vitro. We used spleens as the source of T cells because they are the
closest mouse source to human PBL (the ones generally tested in
vaccinated patients) that gave us the required number of cells for this
assay. Unlike standard in vitro assays, this short period is
insufficient to allow naive T cells to become producers of IFN- or
to allow cell division and is thus likely to be a fairly accurate
measure of the number and/or frequency of currently active
vaccine-induced tumor-specific cells.
With this system, we found that 1) effective in vivo antitumor immunity
can indeed be manipulated by changing the dose, incidence, or route of
vaccination; 2) regardless of the route, frequency, or dose, and
despite great variability in the responses of individual animals to any
particular vaccination protocol, the ex vivo production of IFN-, as
measured by qRT-PCR after a 2-h Ag pulse, correlated very well with the
in vivo anti-tumor response; and 3) low responses, though easily
detectable ex vivo, were often not sufficient to clear the tumor.
The single most important feature of this study was the finding that
the simple presence of a measurable immune response was not always
sufficient to clear a tumor, but that the strength of the response was
critical. Tumor outgrowth in the presence of immunity is not a new
finding. For example, tumor-infiltrating lymphocytes can be isolated
from growing tumors and shown to have anti-tumor specificity;
tumor-specific immunity can be shown in vaccinated patients, though
their tumors continue to grow and many vaccine strategies have failed
to clear tumors, though immunity was clearly established. These
failures of immunity have been variously attributed to outgrowth by
tumor Ag or MHC Ag loss variants (27), to tumor expression
of immune response inhibitory cytokines like TGF- (28)
or apoptotic signals like Fas ligand (29), and to the
establishment of a resistant tumor microenvironment (30).
However, in our study, the instability in tumor Ag expression by the
tumor was minimized and none of the other factors seemed to be major
problems. Although we allowed the tumor to establish itself before
vaccinating, we did not find that it was able to establish a protected
microenvironment in 3 days. Instead, we saw that, if the immune
response was strong enough, as measured by ex vivo production of
IFN- the tumors were almost invariably cleared.
The relationship between strength of response and its efficacy at rejecting a tissue has been known for some time in transplant situations, where grafts that differ by a "weak" minor histocompatibility Ag can take a very long time to reject and are often accepted by a proportion of recipients, even though these recipients clearly become immunized and are able to rapidly reject subsequent grafts (31, 32). In these cases, small grafts are rejected more often than large ones. In the case of a rapidly growing tumor, a weak immune response, though measurable ex vivo, would simply not be able to catch up.
Thus, it is clear that any vaccine programs designed to treat tumors should not rely simply on measurements of the presence or absence of vaccine-elicited immunity. They must take into account the strength of the elicited immune response in vivo and design the vaccination route and schedule to bring the response up to an effective level. This requires assays that accurately measure the strength of an ongoing response, but the majority of assays in current use do not do that. There are three major classes of such assays: 1) those that measure the number of cells that can bind to a particular Ag (such as MHC/Ag tetramer binding assays). These assays, while useful in determining the number of Ag-specific cells, may or may not reflect the functional status of those cells, nor do they measure the number of cells able to recognize other Ags on a tumor; 2) assays that measure the precursor frequency of Ag-specific memory cells for a particular function (such as limiting dilution CTL assays). These types of assays, with their long culture periods, are a measure of clonable memory cells that do not give a clear picture of the currently ongoing activity state of the responding cells in vivo; and 3) assays that attempt to measure the level of currently active lymphocytes immediately ex vivo (such as short-term intracellular cytokine assays, short-term ELISPOT assays, and short-term qRT-PCR). These latter assays have the potential to represent, reasonably accurately, the ongoing in vivo response and therefore can give the type of information needed to determine whether the elicited response is strong enough or needs to be enhanced. Although in this study we did not compare the real-time PCR-based assessment of immune response with other methods, we have previously noted a good correlation with results obtained in clinical settings using tetrameric HLA/epitope complexes and intracellular cytokine staining (15).
Although the production of IFN- correlated well with tumor clearance
in this study and an IFN--dependent mechanism has also been shown to
be effective against the B16 melanoma cells (33), there
are several reasons why assays of other cytokines may be best in other
situations. First, some tissues are exquisitely sensitive to Th1
cytokines such as TNF- and IFN- like pancreatic islets
(34), placenta (35), and eye
(36), whereas others are more resistant. Thus, a
vaccine-elicited IFN- response may be entirely appropriate to clear
some tumors, but useless against others. Second, though IFN- and CTL
often occur concomitantly, there is evidence that the two can also
occur independently (37). For tumors where a CTL response
is necessary, measurement of IFN- may give an accurate assessment in
some patients and not in others. In these cases, direct measurement of
the killing function, or of granzyme or perforin, might be more
appropriate. In the present study, we found that the levels of these
molecules were indeed higher in vaccinated than in control animals.
However, they did not increase in a 2-h stimulus with Ag or with tumor,
suggesting that effector cells control their release by other,
posttranscriptional, means. Thus, it may be necessary to find the best
correlate for each vaccine protocol and each type of tumor. In each
case, it will be important to insure that the assay measures the
strength of the ongoing in vivo response rather than its mere
presence.
Our study shows that, in principle in mice, the strength of a
vaccine-elicited response is critical to its efficacy in clearing a
tumor, that it can be manipulated by varying the dose, frequency and
route of vaccination, and that it can be accurately measured by an
immediate ex vivo method. In light of these data, we re-examined our
earlier study on vaccinated human patients. Though the overall
correlation between tumor regression and ex vivo response was low, we
did find that there was tumor regression and/or stasis in the small
number of patients who made very strong IFN- responses. In addition,
data from various immunization methods tested at our institution
suggest that the vaccination schedule associated with the highest
success rate in inducing ex vivo immune responses is also the most
effective clinically (Ref. 38 and unpublished data). Thus,
although at the individual patient level it might be difficult to
directly relate the intensity of immune response to clinical outcome,
larger population-based studies may yield information comparable to the
mouse model. Inaccuracy in the correlation between in vitro immunity
and clinical effects in human studies may reflect different suitability
of tumor cells as targets for effector T cells as tumors are quite
heterogenous in HLA and/or tumor Ag expression (27).
Finally, the timing in which immune responses have been documented in
human studies has been quite arbitrary, as knowledge of the kinetics of
vaccine-induced cellular responses in humans has not been
comprehensively studied.
Recently, we followed the kinetics of TCR use in response to repeated epitope-specific vaccination in a limited cohort of patients. This preliminary study suggested that increasing the number of vaccinations broadens and intensifies the extent of the immune response (39). Based on this human study and the findings described in this mouse model, we have begun a trial in which patients with advanced cancer are repeatedly boosted to test whether 1) higher precursor frequency of cancer-specific T cells can be generated and 2) improved clinical outcome will follow this enhancement of the immune response.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: -gal, -galactosidase; qRT-PCR, quantitative RT-PCR; FDG, fluorescein di--galactopyranoside; VV, -gal expressing recombinant vaccinia virus; FPV, -gal expressing recombinant fowl pox virus; cVV, control recombinant vaccinia virus; cFPV, control recombinant fowl pox virus; Ct, cycle threshold; Flu, peptide from influenza.
Received for publication July 23, 2001. Accepted for publication October 22, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-D-galactosidase activity after transduction of Escherichia coli LacZ. Proc. Natl. Acad. Sci. USA 85:2603.
in CD8 T cell-mediated immune responses against B16.F10 melanoma cells expressing a viral antigen. Eur. J. Immunol. 30:2507.[Medline]
and tumor necrosis factor-: cytotoxicity to murine islets of Langerhans. J. Immunol. 141:2325.[Abstract]
and TNF- associated with pregnancy outcomes. J. Immunol. 160:2523. in the retina develop inflammation of the eye and photoreceptor loss. Invest Ophthalmol. Visual Sci. 35:2667.This article has been cited by other articles:
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F. Perosa, E. Favoino, C. Vicenti, F. Merchionne, and F. Dammacco Identification of an Antigenic and Immunogenic Motif Expressed by Two 7-Mer Rituximab-Specific Cyclic Peptide Mimotopes: Implication for Peptide-Based Active Immunotherapy J. Immunol., December 1, 2007; 179(11): 7967 - 7974. [Abstract] [Full Text] [PDF]
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 K. M. Zirlik, D. Zahrieh, D. Neuberg, and J. G. Gribben Cytotoxic T cells generated against heteroclitic peptides kill primary tumor cells independent of the binding affinity of the native tumor antigen peptide Blood, December 1, 2006; 108(12): 3865 - 3870. [Abstract] [Full Text] [PDF]
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J. N. Radcliffe, J. S. Roddick, P. S. Friedmann, F. K. Stevenson, and S. M. Thirdborough Prime-Boost with Alternating DNA Vaccines Designed to Engage Different Antigen Presentation Pathways Generates High Frequencies of Peptide-Specific CD8+ T Cells J. Immunol., November 15, 2006; 177(10): 6626 - 6633. [Abstract] [Full Text] [PDF]
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A. Rosato, A. Zoso, S. D. Santa, G. Milan, P. Del Bianco, G. L. De Salvo, and P. Zanovello Predicting Tumor Outcome following Cancer Vaccination by Monitoring Quantitative and Qualitative CD8+ T Cell Parameters J. Immunol., February 1, 2006; 176(3): 1999 - 2006. [Abstract] [Full Text] [PDF]
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 L. A. Emens and E. M. Jaffee Leveraging the Activity of Tumor Vaccines with Cytotoxic Chemotherapy Cancer Res., September 15, 2005; 65(18): 8059 - 8064. [Abstract] [Full Text] [PDF]
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W. Luo, J. C.-f. Hsu, C.-Y. Tsao, E. Ko, X. Wang, and S. Ferrone Differential Immunogenicity of Two Peptides Isolated by High Molecular Weight-Melanoma-Associated Antigen-Specific Monoclonal Antibodies with Different Affinities J. Immunol., June 1, 2005; 174(11): 7104 - 7110. [Abstract] [Full Text] [PDF]
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T. Storni, C. Ruedl, K. Schwarz, R. A. Schwendener, W. A. Renner, and M. F. Bachmann Nonmethylated CG Motifs Packaged into Virus-Like Particles Induce Protective Cytotoxic T Cell Responses in the Absence of Systemic Side Effects J. Immunol., February 1, 2004; 172(3): 1777 - 1785. [Abstract] [Full Text] [PDF]
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A. Rosato, A. Zoso, G. Milan, B. Macino, S. Dalla Santa, V. Tosello, E. Di Carlo, P. Musiani, R. G. Whalen, and P. Zanovello Individual Analysis of Mice Vaccinated against a Weakly Immunogenic Self Tumor-Specific Antigen Reveals a Correlation between CD8 T Cell Response and Antitumor Efficacy J. Immunol., November 15, 2003; 171(10): 5172 - 5179. [Abstract] [Full Text] [PDF]
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 J. W. Smith II, E. B. Walker, B. A. Fox, D. Haley, K. P. Wisner, T. Doran, B. Fisher, L. Justice, W. Wood, J. Vetto, et al. Adjuvant Immunization of HLA-A2-Positive Melanoma Patients With a Modified gp100 Peptide Induces Peptide-Specific CD8+ T-Cell Responses J. Clin. Oncol., April 15, 2003; 21(8): 1562 - 1573. [Abstract] [Full Text] [PDF]
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R. C. Kennedy, M. H. Shearer, A. M. Watts, and R. K. Bright CD4+ T Lymphocytes Play a Critical Role in Antibody Production and Tumor Immunity against Simian Virus 40 Large Tumor Antigen Cancer Res., March 1, 2003; 63(5): 1040 - 1045. [Abstract] [Full Text] [PDF]
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M.-C. Meunier, G. Roy-Proulx, N. Labrecque, and C. Perreault Tissue distribution of target antigen has a decisive influence on the outcome of adoptive cancer immunotherapy Blood, January 15, 2003; 101(2): 766 - 770. [Abstract] [Full Text] [PDF]
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H. C. Hill, T. F. Conway Jr., M. S. Sabel, Y. S. Jong, E. Mathiowitz, R. B. Bankert, and N. K. Egilmez Cancer Immunotherapy with Interleukin 12 and Granulocyte-Macrophage Colony-stimulating Factor-encapsulated Microspheres: Coinduction of Innate and Adaptive Antitumor Immunity and Cure of Disseminated Disease Cancer Res., December 15, 2002; 62(24): 7254 - 7263. [Abstract] [Full Text] [PDF]
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J. Rice, S. Buchan, and F. K. Stevenson Critical Components of a DNA Fusion Vaccine Able to Induce Protective Cytotoxic T Cells Against a Single Epitope of a Tumor Antigen J. Immunol., October 1, 2002; 169(7): 3908 - 3913. [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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