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*
Department of Transfusion Medicine, Clinical Center,
Surgery Branch, National Cancer Institute,
Biostatistics and Data Management Section, National Cancer Institute, and
§
Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, 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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Therefore, the paradoxical behavior of vaccine-elicited immune responses questions, among other variables, the validity of data obtained by ex vivo stimulation and expansion of T cells. These assays require repeated stimulation with arbitrarily chosen concentrations of exogenous epitopes and cytokines, which could alter the functional and phenotypic characteristics of the T cells. Therefore, other assays directly measuring T cell reactivity in PBMC have been fashioned. Among them, the enzyme-linked immunospot (ELISPOT) assay can detect MA-specific CTL directly in PBMC (6) but may overlook inactive epitope-specific T cells that are unable to respond to epitope-specific stimulation. Tetrameric HLA/peptide complexes (tHLA) allow for direct measurement of epitope-specific T cell precursor frequency (TCPF) without in vitro manipulation (7). This method has been successfully used to measure T cell responses to viral infections (8, 9) and has been shown to estimate higher TCPF in PBMC than other assays (10). Furthermore, as this method does not rely on functional T cell responses, supplementary information to functional assays can be obtained.
We analyzed g209- and g209–2M-specific TCPF in pre- and posttreatment PBMC from 23 melanoma patients vaccinated with g209–2M peptide emulsified in IFA. We also evaluated whether enhancement in TCPF secondary to vaccination corresponded to increased susceptibility to in vitro stimulation with the relevant epitope. Furthermore, as the frequency of MA-specific T cells at tumor site might be of greater relevance than in PBMC, we compared, when available, g209-specific TCPF in PBMC and tumor infiltrating lymphocyte (TIL) pairs simultaneously obtained after vaccination.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HLA-A*0201 patients received the g209–2M peptide in IFA. Representative PBMC were obtained from patients treated with peptide in IFA (n = 7, P1–P7), peptide with IL-12 s.c. (IL-12, n = 5, P8–P12), or with high dose (720,000 IU/kg every 8 h) IL-2 i.v. (IL-2, n = 11, P13-P23). These PBMC were selected according to previous in vitro sensitization, suggesting different vaccination outcomes in relation to concomitant cytokine treatment (2). Postvaccination PBMC from the patients treated with peptide alone or peptide with IL-12 had demonstrated tumor specificity after in vitro expansion, whereas PBMC from patients who had been treated with peptide and IL-2 did not. Vaccinations were administered at 3-wk intervals, and blood samples were obtained three weeks after vaccination unless otherwise specified. The HLA class I phenotype of patients was determined on PBMC using sequence specific primer-PCR (11). PCR was also used for molecular subtyping of HLA-A2 (12).
Cells and cultures
Samples were obtained from blood draws and leukapheresis of melanoma patients before and after vaccination with g209–2M peptide. PBMC were isolated by Ficoll gradient separation and frozen until analysis. For analysis of TCPF in TIL, excised tumor samples were enzymatically digested and frozen without separation of mononuclear cells from tumor cells as previously described (13). Analysis of MA-specific T cells was performed after overnight resting of thawed PBMC in complete medium consisting of RPMI 1640 medium (Biofluids, Rockville, MD) supplemented with 10 mM HEPES buffer, 100 U/ml penicillin-streptomycin (Biofluids), 10 µg/ml ciprofloxacin (Bayer, West Haven, CT), 0.03% L-glutamine (Biofluids), 0.5 mg/ml amphotericin B (Biofluids), 10% heat-inactivated human AB serum (Gemini Bioproducts, Calabasas, CA), and 300 IU IL-2/ml. This procedure allowed depletion of adherent monocytes. PBMC were also analyzed after 10 days of in vitro culture following stimulation with exogenous peptide. This was achieved by the administration of 1 µM peptide in complete medium to the PBMC at the time of thaw and the addition of IL-2 (300 IU/ml) the following day and every third day thereafter.
Epitope-specific T cell staining using HLA-A2 tetramer
Tetrameric peptide-HLA-A*0201 complexes were produced 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: gp100:209–217 (ITDQVTCPFSV, g209); gp100:209–217 (210M) (IMDQVTCPFSV, g209–2M); and FluM1:58–66 (GILGFVFTL, Flu). 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 1 x 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 then continued for 30 min with 10 µl anti-CD8 mAb (100 µg/ml; Becton Dickinson, San Jose, CA). Cells were washed twice in 2-ml cold FACS buffer before analysis by FACS (Becton Dickinson). Fifty thousand events were acquired for CTL cultures and 200,000 events for PBL samples. Staining of tumor preparations was performed similarly to the preparation of PBMC with overnight resting in complete medium and direct staining of nonadherent cells.
FACS analysis for intracellular expression of IFN-
Nonadherent PBMC (1 x 106 cells)
were stimulated for 6 h with peptide (1 µg/ml) pulsed T2 cells
(1 x 106 cell/ml). After 2 h,
Brefeldin A (10 µg/ml) (Sigma, Deisenhofen, Germany) was added. After
four additional hours, the cells were treated with 4500 U DNase I
(Calbiochem, La Jolla, CA) for 5 min at 37°C. EDTA (0.1 M) was added
to each well before washing with cold PBS. Cells were fixed with 4%
paraformaldehyde for 5 min and washed in PBS containing 0.1% BSA.
Cells were blocked overnight with PBS/5% milk on ice in cold room.
Permeabilization of cells was performed with PBS. After staining with
mAb for 30 min on ice, cells were washed in PBS. Staining with
tetramers was performed before fixation of cells. The rest of the
procedure was conducted according to the same protocol as all other
stains. All samples were analyzed on a Becton Dickinson FACSCalibur
flow cytometer using the CellQuest software. Live-gating on
lymphocytes, CD3+, and CD8+
was performed during acquisition. The following mAbs were used:
allophyocyanin-conjugated mouse anti-human CD3 (IgG1),
peridinin-chlorophyll protein-conjugated mouse anti-human CD8
(IgG1), and fluorescein (FITC)-conjugated mouse anti-human CD45RA
were purchased from Becton Dickinson (Heidelberg, Germany), and
fluorescein (FITC)-conjugated mouse anti-human IFN- (IgG1) and
fluorescein (FITC)-conjugated mouse anti-human CD45RO (PharMingen,
San Diego, CA).
Statistical analysis
In the light scatter, the lymphocyte population was gated in for evaluation. The frequency (f) of peptide-specific T cells per 106 CD8+ cells was calculated using the following formula: f = URQ/(URQ + LRQ) x 106 CD8+ cells, with URQ containing the tHLA+, CD8+ cells and LRQ containing all other CD8+ cells. From these frequencies, the background with CD8+ staining only was subtracted for each sample to obtain the corrected frequency (fc). The fc is presented as the number of peptide-specific T cells per 1 x 106 CD8+ T cells.
For statistical comparison, the basic unit of analysis was the log10 of change between posttreatment vs pretreatment fc for each day, staining, and stimulation condition. The log10 was chosen because of the wide range of fc values observed in different patients. Because of varying intervals between pre- and posttreatment samples, Spearman correlation analyses and scatter plots were constructed to determine whether there was a relationship between interval (days) from pretreatment to posttreatment and log10 change in fc for each experimental condition.
For each condition, the statistical significance of the log10 change from pre- to posttreatment fc was determined by the Wilcoxon signed rank test, separately for each treatment group. In addition, specific analyses comparing changes under one experimental condition to changes with another condition were also done by the Wilcoxon signed rank test after subtracting one change [log10 (postpre)] from the other.
Finally, the Kruskal-Wallis (KW) test was used to determine the significance of the difference among the three treatment groups with respect to any of the changes or comparisons of the changes from pre- to posttreatment within a particular condition. These p values are indicated as KW in the figures. All p values are two sided and have not been explicitly adjusted for multiple comparisons because all analyses are being done on small groups of patients and with exploratory intent; thus, the results should be interpreted as hypothesis generating until confirmed by other studies.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The TCPF of g209- and g209–2M-specific CD8+
cells were determined in PBMC by g209 and g209–2M tHLA staining as
shown in the patient’s sample illustrated in Fig. 1
. The precursor frequencies calculated
(fc) were then calculated as
shown in Table I. Detection of
Flu-specific CD8+ cells was included as a
control. Because of varying intervals, ranging from 40 to 283 days,
between pre- and posttreatment PBMC samples, the effect of the time was
assessed by Spearman correlation analysis. The results of the
correlation analysis suggested that slight variations in
fc could be considered a negligible
factor in the interpretation of the results (data not shown). The range
of fc noted was from undetectable
above background, particularly in the peptide + IL-2 group to
14,806/106 CD8+ cells for
g209 (P4, postvaccination), 24,576/106
CD8+ cells for g209–2M (P12, postvaccination),
and 21,583/106 CD8+ cells
for Flu (P12, prevaccination). The variability of the results and the
details for patients in the various cohorts are shown in Table I.
Statistical comparison of the pre- and postvaccination
fc for each patient in the peptide
alone (P alone) treatment group showed a significant increase of g209-
and g209–2M-specific CD8+ cells (g209: mean
log10 difference (
) = 0.73,
p = 0.016; g209–2M: = 0.61, p
= 0.016) (Figs. 2A and
3A). A similar change was
observed in the peptide + IL-12 (P + IL-12) treatment group (g209:
= 0.60, p = 0.125; g209–2M: = 0.78,
p = 0.0625). The peptide + IL-2 group (P + IL-2) did
not show any obvious trends (g209: = -0.07, p
= 0.240; g209–2M: = -0.19, p = 0.17). The
differences of trends among various groups were statistically
significant (g209: KW = 0.018; g209–2M: KW = 0.0024). Flu
fc values were not significantly
different between pre- and postvaccination PBMC samples in all
treatment groups (P alone: = -0.22, p = 0.30;
P + IL-12: = 0.07, p = 0.44; P + IL-2:
= -0.58, p = 0.28) (Fig. 4A). Thus,
vaccination-dependent enhancement of TCPF could be detected in peptide
treated patients who had not received i.v. IL-2. Sample collection in
this study was dictated by the clinical protocol structure, which
provided for patient return to our institution for re-evaluation and
vaccine administration at 3-wk intervals. However, to investigate
whether the relatively low TCPF observed were due to the distance
between the last vaccination and sample collection, we tested
patients’ PBMC at shorter intervals after vaccine administration.
Samples were collected before and 3, 7, 10, and 14 days after
vaccination. At no time point were TCPF fluctuations noted (data not
shown). Thus, it is unlikely that significantly higher frequencies were
missed in the cohort of patients studied because of the delay in which
the blood samples had been collected.
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Because determination of vaccine-specific TCPF does not yield
information about their ability to respond to epitope-specific
stimulation, we performed an in vitro expansion of the same PBMC in all
23 patients as exemplified for patient 6 (Fig. 5). The selection of PBMC as APC
emphasizes a bias toward expansion of memory T cells elicited by the
vaccination. Furthermore, this method was selected because it is the
one previously described for the assessment of response to vaccination.
Thus, analysis of CTL expansion after in vitro stimulation using tHLA
can be directly compared with results previously reported by our group
obtained by evaluating cytokine release and cytotoxicity of
vaccine-induced in vitro-expanded T cells (2, 3, 4). As shown
in this patient, an enrichment of vaccine-specific T cells could be
easily demonstrated after in vitro stimulation of post- but not
prevaccination PBMC. Concomitant stimulation of T cells specific for an
epitope irrelevant to the one used for in vitro stimulation was
occasionally noted. In patient 6, we noted persistence and/or minor
expansion of Flu-specific CTL in cultures stimulated with g209–2M.
Conversely, g209 and g209–2M-specific T cells could be identified in
postvaccination cultures stimulated with Flu. In no case could the
concomitant T cell expansion be noted in unstimulated control cultures
(data not shown).
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). The increase was
less pronounced in the P + IL-2 group. In fact, most patients in the P
+ IL-2 group did not show any increase. The amplified
fc differences between pre- and
postvaccination cultures underlined the trends observed in PBMC within
each treatment group. However, fc
differences in pre- and postvaccination PBMC of single patients were
not reflected quantitatively by fc
differences in the corresponding in vitro cultures.
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, B and C;
Fig. 3, B and C, and Fig. 4B). The P +
IL-12 group demonstrated the same trend but did not reach statistical
significance. Measurement of vaccine-specific T cell expansion in
patients receiving peptide plus IL-2 also revealed a modest but
significant increase in post- compared with prevaccination samples.
Thus, it appears that the inability to detect an enhancement of
vaccine-specific T cell in these patients was in part due to a
limitation in the sensitivity of tHLA staining rather than an absolute
lack of effect of the vaccine. This analysis, therefore, demonstrates
that the enhancement of TCPF detected in PBMC in response to
vaccination is associated with an enhanced susceptibility to in vitro
epitope-specific stimulation.
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In one patient (P4), TCPF was monitored throughout treatment with
multiple vaccinations (before and after 1, 2, 4, 6, 8, and 10
vaccinations) (Fig. 6). One vaccination was
not sufficient to elicit a detectable increase in TCPC. However, a
strong enhancement was noted 3 wk after the second vaccination (Fig. 1)
at which time point there was no evidence of tumor regression. Starting
from the third vaccination, IL-2 was added. Surprisingly, the TCPF
decreased and tumors began to shrink. Thus, the detection of
vaccine-specific T cells in PBMC after two vaccinations did not
correlate with clinical outcome. Rapid expansion in response to
vaccine-specific stimulation in vitro makes it unlikely that functional
unresponsiveness was responsible for tumor progression. To more
directly assess the functional state of g209/g209–2M-specific T cell,
pre- and postvaccination PBMC were compared for intracellular IFN-
expression in response to stimulation with T2 cells exogenously pulsed
with g209 or g209–2M (Fig. 7). This
analysis demonstrated a specific enhancement of g209/g209–2M reactive
T cells only in postvaccination PBMC, which correlated with specific
down-regulation of TCR as judged by tetramer staining. Interestingly
the number of IFN--expressing T cells in the postvaccination PBMC
sample was noted to be roughly half the number of tetramer positive T
cells. It is possible that tetramer staining of postvaccination PBMC
picks up a fraction of vaccine-induced T cells that do not produce
IFN- in response to Ag stimulation in the in vitro conditions
exercised used for this study.
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Comparison of PBMC/tumor pairs
The low TCPF observed in the P + IL-2 group, which has been
associated with clinical responses, may be explained by a migration of
tumor-specific T cells from the systemic circulation to the tumor site.
To address whether this phenomenon really occurs, we analyzed PBMC and
tumor preparations, which had been obtained at the same time point
after vaccination. Seven such pairs were available for analysis (Table V). In two of seven cases
(L.R. and D.W.), a suggestive evidence of localization was seen.
Interestingly, these lesions demonstrated high expression of the target
Ag gp100 by immunocytochemistry. It should be emphasized, however, that
this analysis could be strongly biased by the selection of lesions that
had persisted after immunization.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The identification of MA epitopes recognized by T cells has led to the utilization of minimal peptide sequences for the in vivo induction or amplification of systemic, tumor-specific T cell responses (1, 2). Results from pilot studies testify for the high specificity of the induced T cell responses against HLA-matched tumor cells (2, 3, 4). These studies gave the impression that vaccines induce powerful immunizations comparable to those demonstrable against common pathogens such as the influenza virus to which individuals are repeatedly exposed throughout their lifetime. In most cases, this vaccine-induced T cell reactivity still does not lead to tumor regression.
Most studies thus far have measured T cell response to epitope-specific vaccination by comparative assessment of pre- and postvaccination PBMC for tumor-specific T cell expansion in vitro in response to stimulation with relevant and irrelevant epitopes, as originally described by Vitiello et al. (18) in a viral system. These assays are excellent for qualitative assessment of T cell responses at two time points in an individual’s life or for nonparametric comparison of treatment outcomes in different patient groups. However, they cannot provide quantitative insight about the strength of the observed response because of the arbitrary nature of the stimulus applied ex vivo and the addition of secondary proliferative stimuli, which most commonly consists of IL-2. Thus, as for TIL, it is likely that the immune responses judged after ex vivo expansion of postvaccination PBMC overestimate quantitatively the strength of the immune reaction within the organism.
Therefore, other methods of analysis have been fashioned to evaluate T cell responses directly without the need for ex vivo amplification. Among these, the ELISPOT assay has enjoyed notable popularity because of its simplicity, relative accuracy and sensitivity (19). Analysis of postvaccination PBMC from patients treated with g209–2M peptide in IFA could identify T cells specific for the altered epitope in four of six patients. In the four patients, the estimated frequencies ranged between 1/1000 and 1/2000 epitope-specific T cells (6). In no patient was it possible to identify T cells recognizing the natural epitope (g209) by the ELISPOT assay, whereas limiting dilution assays estimated precursor frequencies for g209-reactive T cells to range between 1/3000 and 1/6000 (2). Because of the dependency upon cytokine secretion/proliferation, these assays may underestimate the actual frequency of CTL precursors by not identifying T cells with a threshold for cytokine expression/proliferation above the stimulus applied in the assays (10). Moreover, naive T cells, less responsive to epitope-specific stimulation, might be missed by these functional assays (20). Recently, the use of HLA/epitope tetramers (7) has offered a tool to directly measure the frequency of CTL precursors presumably independently of their functional state. Measurements by this assay demonstrated CTL precursor frequencies considerably higher than those suggested by ELISPOT or limiting dilution assays (10).
T cell responses to epitope-specific vaccination have not been measured with this direct assay, yet. Several studies have measured the response of T cells to acute or chronic viral infections or during ongoing autoimmune episodes (8, 9, 10, 21, 22, 23). So far studies on tumor reactivity have been presented by few groups of investigators and have been primarily limited to the analysis of MART-1 and tyrosinase. In melanoma patients with vitiligo, MART-1-specific CTL have been identified at a frequency up to 0.67% of CD8+ T cells (24). Furthermore, MART-1-specific T cells could be identified in melanoma infiltrated lymph nodes with a frequency ranging from 0.22 to 1.8% of CD8+ T cells and correlated with MA expression (25). Characterization of circulating T cells demonstrated identifiable MA-specific T cells in approximately half of patients affected with metastatic melanoma (26). Functional characterization of MA-specific T cells from one patient lead to the generalization that some of the T cells identified with tHLA are unresponsive to Ag stimulation and, thus, unable to control tumor growth. This study is the first quantitative evaluation of the response to an HLA class I-restricted epitope vaccination. Epitope-specific vaccination yielded significant differences between the pre- and posttreatment CTL precursor frequencies. Vaccine-specific T cell frequency increased up to 2.5% of CD8+ cells after vaccination, and the frequency of T cell recognizing the natural gp100 epitope (g209) was enhanced up to 1.4%. In a significant proportion of patients, frequency of g209 recognizing T cell after vaccination ranged between 0.2 to 0.9%. Thus, a significant conceptual finding of this study is the limited extent of vaccine-specific response. We were surprised at the relatively low numbers of CTL precursors after vaccination even in patients’ samples that boasted an exceptional epitope-specific expansion in vitro. An inverse correlation has been reported between HIV-specific CTL frequency and viral RNA load in HIV infected individuals (8). Furthermore, TCPF as high as 2% of CD8+ T cells have been reported in HIV-infected patients, who remained asymptomatic (22). Thus, it is possible that the immune response elicited by the vaccination regimen used in this study did not reach the quantitative capacity necessary for tumor regression.
Clonal deletion, exhaustion, or senescence (27, 28, 29, 30, 31, 32, 33, 34) have
been implicated in the induction of systemic, epitope-specific immune
tolerance. However, because g209-specific T cell could be identified
after vaccination in this study, deletion of tumor-reactive T cells may
not be as significant in humans as suggested by preclinical models
(27, 30, 33, 34, 35). Inadequate immune responses in patients
with cancer and other chronic illnesses have been attributed to
decreased TCR signaling capacity (36, 37) or circulating
immune-suppressive cytokines (37). Finally, analysis of
MA-specific T cells from one patient has lead to the generalization
that tumor-specific T cells may be anergic in vivo (26).
However, in this study, T cells elicited by the vaccine could be
readily stimulated with the cognate epitope to rapidly proliferate in
culture. Furthermore, analysis of postvaccination PBMC identified a
significant percentage of vaccine-specific T cells capable to secrete
IFN- in response to vaccine-specific stimulation. Taken together,
these data suggest that the extent rather than the quality of the
response might be the more significant limitation of the vaccination
protocol analyzed in this study.
Although differences in TCPF were detectable between pre- and postvaccination PBMC selected from patients that had been treated with peptide alone, no significant differences could be detected in patients who had received peptide plus systemic IL-2 therapy and had shown no enhanced reactivity by in vitro sensitization assays (2). The association of undetectable vaccine-specific T cells with the enhanced frequency of clinical responses after systemic administration of IL-2 remains mysterious. One possibility is that the responsiveness of T cell to in vitro restimulation might be reduced in patients who received IL-2. This study demonstrates instead that the number rather than susceptibility to in vitro expansion is decreased in these patients. It has been suggested that tumors will induce tolerance by presenting epitope-specific stimulation (signal one) without costimulation (signal two) to wandering MA-recognizing T cells (38). By increasing vascular permeability, IL-2 might facilitate encounters between T cells and cancer cells that lead to reduced tumor burden and T cell number at the same time. Study of seven simultaneously obtained PBMC and tumor samples demonstrated a slightly increased frequency of vaccine-induced T cells at tumor site in only two of the pairs studied in correlation with expression of gp100. It is possible that insufficient localization and/or poor survival of vaccine-induced T cells at tumor site may be the reason why the lesions analyzed in this study did not regress in response to therapy.
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Footnotes |
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2 Abbreviations used in this paper: MA, melanoma Ag; tHLA, tetramer HLA; g209, gp100:209–217; g209–2M, gp100:209–217(210M); ELISPOT, enzyme-linked immunospot; MART-1, MART-1:26–35 (27L); Flu, FluM1:58–66; TCPF, precursor frequency; TIL, tumor infiltrating lymphocyte; fc, calculated frequency; KW, Kruskal-Wallis; P, peptide.
Received for publication July 16, 1999. Accepted for publication September 14, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-ELISPOT assay. Int. J Cancer 71:932.[Medline]
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Y. Nagata, T. Hanagiri, M. Takenoyama, T. Fukuyama, M. Mizukami, T. So, Y. Ichiki, M. Sugaya, K. Sugio, and K. Yasumoto Identification of the HLA-Cw*0702-Restricted Tumor-Associated Antigen Recognized by a CTL Clone from a Lung Cancer Patient Clin. Cancer Res., July 15, 2005; 11(14): 5265 - 5272. [Abstract] [Full Text] [PDF]
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Y. Suzuki, D. Wakita, K. Chamoto, Y. Narita, T. Tsuji, T. Takeshima, H. Gyobu, Y. Kawarada, S. Kondo, S. Akira, et al. Liposome-Encapsulated CpG Oligodeoxynucleotides as a Potent Adjuvant for Inducing Type 1 Innate Immunity Cancer Res., December 1, 2004; 64(23): 8754 - 8760. [Abstract] [Full Text] [PDF]
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V. Monsurro, E. Wang, Y. Yamano, S. A. Migueles, M. C. Panelli, K. Smith, D. Nagorsen, M. Connors, S. Jacobson, and F. M. Marincola Quiescent phenotype of tumor-specific CD8+ T cells following immunization Blood, October 1, 2004; 104(7): 1970 - 1978. [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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H. L. Hanson, S. S. Kang, L. A. Norian, K. Matsui, L. A. O'Mara, and P. M. Allen CD4-Directed Peptide Vaccination Augments an Antitumor Response, but Efficacy Is Limited by the Number of CD8+ T Cell Precursors J. Immunol., April 1, 2004; 172(7): 4215 - 4224. [Abstract] [Full Text] [PDF]
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S. Yang and F. G. Haluska Treatment of Melanoma with 5-Fluorouracil or Dacarbazine In Vitro Sensitizes Cells to Antigen-Specific CTL Lysis through Perforin/Granzyme- and Fas-Mediated Pathways J. Immunol., April 1, 2004; 172(7): 4599 - 4608. [Abstract] [Full Text] [PDF]
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Y. Sugita, H. Wada, S. Fujita, T. Nakata, S. Sato, Y. Noguchi, A. A. Jungbluth, M. Yamaguchi, Y.-T. Chen, E. Stockert, et al. NY-ESO-1 Expression and Immunogenicity in Malignant and Benign Breast Tumors Cancer Res., March 15, 2004; 64(6): 2199 - 2204. [Abstract] [Full Text] [PDF]
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H. Gyobu, T. Tsuji, Y. Suzuki, T. Ohkuri, K. Chamoto, M. Kuroki, H. Miyoshi, Y. Kawarada, H. Katoh, T. Takeshima, et al. Generation and Targeting of Human Tumor-Specific Tc1 and Th1 Cells Transduced with a Lentivirus Containing a Chimeric Immunoglobulin T-Cell Receptor Cancer Res., February 15, 2004; 64(4): 1490 - 1495. [Abstract] [Full Text] [PDF]
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K. Teramoto, K. Kontani, Y. Ozaki, S. Sawai, N. Tezuka, T. Nagata, S. Fujino, Y. Itoh, O. Taguchi, Y. Koide, et al. Deoxyribonucleic Acid (DNA) Encoding a Pan-Major Histocompatibility Complex Class II Peptide Analogue Augmented Antigen-specific Cellular Immunity and Suppressive Effects on Tumor Growth Elicited by DNA Vaccine Immunotherapy Cancer Res., November 15, 2003; 63(22): 7920 - 7925. [Abstract] [Full Text] [PDF]
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D. Nagorsen, C. Scheibenbogen, F. M. Marincola, A. Letsch, and U. Keilholz Natural T Cell Immunity against Cancer Clin. Cancer Res., October 1, 2003; 9(12): 4296 - 4303. [Abstract] [Full Text] [PDF]
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 M. Sato, K. Chamoto, and T. Nishimura A novel tumor-vaccine cell therapy using bone marrow-derived dendritic cell type 1 and antigen-specific Th1 cells Int. Immunol., July 1, 2003; 15(7): 837 - 843. [Abstract] [Full Text] [PDF]
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 G. Parmiani, L. Pilla, C. Castelli, and L. Rivoltini Vaccination of patients with solid tumours Ann. Onc., June 1, 2003; 14(6): 817 - 824. [Abstract] [Full Text] [PDF]
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A. Rosato, S. D. Santa, A. Zoso, S. Giacomelli, G. Milan, B. Macino, V. Tosello, P. Dellabona, P.-L. Lollini, C. De Giovanni, et al. The Cytotoxic T-Lymphocyte Response against a Poorly Immunogenic Mammary Adenocarcinoma Is Focused on a Single Immunodominant Class I Epitope Derived from the gp70 Env Product of an Endogenous Retrovirus Cancer Res., May 1, 2003; 63(9): 2158 - 2163. [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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L. H. Butterfield, A. Ribas, V. B. Dissette, S. N. Amarnani, H. T. Vu, D. Oseguera, H.-J. Wang, R. M. Elashoff, W. H. McBride, B. Mukherji, et al. Determinant Spreading Associated with Clinical Response in Dendritic Cell-based Immunotherapy for Malignant Melanoma Clin. Cancer Res., March 1, 2003; 9(3): 998 - 1008. [Abstract] [Full Text] [PDF]
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N. Meidenbauer, J. Marienhagen, M. Laumer, S. Vogl, J. Heymann, R. Andreesen, and A. Mackensen Survival and Tumor Localization of Adoptively Transferred Melan-A-Specific T Cells in Melanoma Patients J. Immunol., February 15, 2003; 170(4): 2161 - 2169. [Abstract] [Full Text] [PDF]
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I. D. Davis, M. Jefford, P. Parente, and J. Cebon Rational approaches to human cancer immunotherapy J. Leukoc. Biol., January 1, 2003; 73(1): 3 - 29. [Abstract] [Full Text] [PDF]
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L.-X. Wang, B.-G. Chen, and G. E. Plautz Adoptive Immunotherapy of Advanced Tumors with CD62 L-Selectinlow Tumor-Sensitized T Lymphocytes Following Ex Vivo Hyperexpansion J. Immunol., September 15, 2002; 169(6): 3314 - 3320. [Abstract] [Full Text] [PDF]
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H.-M. Hu, C. H. Poehlein, W. J. Urba, and B. A. Fox Development of Antitumor Immune Responses in Reconstituted Lymphopenic Hosts Cancer Res., July 15, 2002; 62(14): 3914 - 3919. [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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S. G. Schaed, V. M. Klimek, K. S. Panageas, C. M. Musselli, L. Butterworth, W.-J. Hwu, P. O. Livingston, L. Williams, J. J. Lewis, A. N. Houghton, et al. T-Cell Responses against Tyrosinase 368-376(370D) Peptide in HLA*A0201+ Melanoma Patients: Randomized Trial Comparing Incomplete Freund's Adjuvant, Granulocyte Macrophage Colony-stimulating Factor, and QS-21 as Immunological Adjuvants Clin. Cancer Res., May 1, 2002; 8(5): 967 - 972. [Abstract] [Full Text] [PDF]
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D. Valmori, V. Dutoit, V. Schnuriger, A.-L. Quiquerez, M. J. Pittet, P. Guillaume, V. Rubio-Godoy, P. R. Walker, D. Rimoldi, D. Lienard, et al. Vaccination with a Melan-A Peptide Selects an Oligoclonal T Cell Population with Increased Functional Avidity and Tumor Reactivity J. Immunol., April 15, 2002; 168(8): 4231 - 4240. [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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J. D. Geiger, R. J. Hutchinson, L. F. Hohenkirk, E. A. McKenna, G. A. Yanik, J. E. Levine, A. E. Chang, T. M. Braun, and J. J. Mule Vaccination Of Pediatric Solid Tumor Patients with Tumor Lysate-pulsed Dendritic Cells Can Expand Specific T Cells and Mediate Tumor Regression Cancer Res., December 1, 2001; 61(23): 8513 - 8519. [Abstract] [Full Text] [PDF]
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J. Kjaergaard, L. Peng, P. A. Cohen, J. A. Drazba, A. D. Weinberg, and S. Shu Augmentation Versus Inhibition: Effects of Conjunctional OX-40 Receptor Monoclonal Antibody and IL-2 Treatment on Adoptive Immunotherapy of Advanced Tumor J. Immunol., December 1, 2001; 167(11): 6669 - 6677. [Abstract] [Full Text] [PDF]
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C.-L. Tso, A. Zisman, A. Pantuck, R. Calilliw, J. M. Hernandez, S. Paik, D. Nguyen, B. Gitlitz, P. I. Shintaku, J. de Kernion, et al. Induction of G250-targeted and T-Cell-mediated Antitumor Activity against Renal Cell Carcinoma Using a Chimeric Fusion Protein Consisting of G250 and Granulocyte/Monocyte-Colony Stimulating Factor Cancer Res., November 1, 2001; 61(21): 7925 - 7933. [Abstract] [Full Text] [PDF]
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P. Lee, F. Wang, J. Kuniyoshi, V. Rubio, T. Stuges, S. Groshen, C. Gee, R. Lau, G. Jeffery, K. Margolin, et al. Effects of Interleukin-12 on the Immune Response to a Multipeptide Vaccine for Resected Metastatic Melanoma J. Clin. Oncol., September 15, 2001; 19(18): 3836 - 3847. [Abstract] [Full Text] [PDF]
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J. T. Kurnick, T. Ramirez-Montagut, L. A. Boyle, D. M. Andrews, F. Pandolfi, P. J. Durda, D. Butera, I. S. Dunn, E. M. Benson, S. J. P. Gobin, et al. A Novel Autocrine Pathway of Tumor Escape from Immune Recognition: Melanoma Cell Lines Produce a Soluble Protein That Diminishes Expression of the Gene Encoding the Melanocyte Lineage Melan-A/MART-1 Antigen Through Down-Modulation of Its Promoter J. Immunol., August 1, 2001; 167(3): 1204 - 1211. [Abstract] [Full Text] [PDF]
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S. Weijzen, S. C. Meredith, M. P. Velders, A. G. Elmishad, H. Schreiber, and W. M. Kast Pharmacokinetic Differences Between a T Cell-Tolerizing and a T Cell-Activating Peptide J. Immunol., June 15, 2001; 166(12): 7151 - 7157. [Abstract] [Full Text] [PDF]
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M. J. Pittet, A. Zippelius, D. E. Speiser, M. Assenmacher, P. Guillaume, D. Valmori, D. Lienard, F. Lejeune, J.-C. Cerottini, and P. Romero Ex Vivo IFN-{{gamma}} Secretion by Circulating CD8 T Lymphocytes: Implications of a Novel Approach for T Cell Monitoring in Infectious and Malignant Diseases J. Immunol., June 15, 2001; 166(12): 7634 - 7640. [Abstract] [Full Text] [PDF]
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D. W. Ju, Q. Tao, G. Lou, M. Bai, L. He, Y. Yang, and X. Cao Interleukin 18 Transfection Enhances Antitumor Immunity Induced by Dendritic Cell-Tumor Cell Conjugates Cancer Res., May 1, 2001; 61(9): 3735 - 3740. [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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 W. C. To, B. G. Wood, J. C. Krauss, M. Strome, R. M. Esclamado, P. Lavertu, D. Dasko, J. A. Kim, G. E. Plautz, B. E. Leff, et al. Systemic Adoptive T-Cell Immunotherapy in Recurrent and Metastatic Carcinoma of the Head and Neck: A Phase 1 Study Arch Otolaryngol Head Neck Surg, October 1, 2000; 126(10): 1225 - 1231. [Abstract] [Full Text] [PDF]
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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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U. S. Kammula, K.-H. Lee, A. I Riker, E. Wang, G. A. Ohnmacht, S. A. Rosenberg, and F. M. Marincola Functional Analysis of Antigen-Specific T Lymphocytes by Serial Measurement of Gene Expression in Peripheral Blood Mononuclear Cells and Tumor Specimens J. Immunol., December 15, 1999; 163(12): 6867 - 6875. [Abstract] [Full Text] [PDF]
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