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* Immunogenetics Section, Department of Transfusion Medicine, Clinical Center, and
Surgery Branch, 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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Because tHLA-based phenotyping of T cells does not yield information
about their effector function, it has been postulated that the lack of
function of tumor Ag-specific T cells could be due to a suboptimal
status of activation (5). Pittet et al. (6)
noted that tumor Ag-specific T cells in patients with melanoma are,
contrary to those analyzed in healthy individuals,
CD45RAlow and can secrete IFN-
upon cognate
stimulation compatible with a functional differentiation into competent
effector/memory T cells in vivo. We have previously argued that
vaccine-induced T cells should be at least partially functional based
on the fact that they can express IFN- mRNA and protein ex vivo upon
cognate stimulation (7). Stimulation with vaccine-related
epitopes induced IFN- expression detectable by intracellular
cytokine analysis and quantitative real-time PCR, suggesting that
vaccine-elicited CTL retain Ag responsiveness (4).
However, recent studies suggest that IFN- expression by T cells may
not be the functional parameter that might best describe their
potential as effector cells. Appay et al. (8) observed in
patients with chronic HIV infection that CMV-specific differ from
HIV-specific circulating CD8+ T cells because
they express significantly lower levels of perforin despite retaining
the ability to secrete IFN- in response to epitope stimulation. In
addition, these lymphocytes retained the expression of CD27, which was
interpreted by the authors as a sign of impaired maturation toward
fully developed effector cells. It is therefore possible that
Ag-experienced vaccine-induced T cells may be in an incomplete stage of
differentiation, which explains their limited effect on tumor
growth. Functionally distinct phenotypes of
CD8+ T cells spanning from a naive to an effector
and/or memory stage of differentiation have been described by several
groups (9, 10). According to Hamman et al.
(9), memory-type CD8+ T cells
are CD45RA-CD27+, can
express a broad range of cytokines (IL-2, IFN-, TNF-
, and IL-4)
in response to cognate stimulation, and do not display cytolytic
activity without prior in vitro stimulation (IVS). In contrast,
effector T cells have a
CD45RA+C27- phenotype,
release a more stringent array of cytokines upon stimulation (IFN-
and TNF-), and display strong cytolytic activity with high
expression of perforin and granzyme without the need for in vitro
prestimulation. Thus, combining phenotypic with functional proprieties,
the authors suggested four distinct populations of
CD8+ T cells depending on their CD45RA and CD27
expression levels: naive T cells
(CD45RA+CD27+), effector T
cells (CD45RA+CD27-),
memory T cells
(CD45RA-CD27+), and
effector/memory T cells
(CD45RA-CD27-). This
classification has been extensively used subsequently in the context of
various infectious diseases and other immune pathologies
(11, 12, 13).
Independent of their intrinsic status of activation and/or
differentiation, circulating lymphocytes need to be able to localize in
target organs to exert their effector function. It has been suggested
that two subsets of memory T lymphocytes can be identified with
distinct homing potentials and effector functions based on their level
of expression of the chemokine receptor CCR7 (14).
CCR7- memory cells express receptors for
migration to target tissues and are ready to exert their effector
function. Conversely, CCR7+ memory cells express
lymph node-homing receptors and lack immediate effector function.
Champagne et al. (15) suggested that
CD8+ T lymphocytes follow a consistent
differentiation pattern that flows from
CD45RA+CCR7+
CD45RA-CCR7+CD45RA-CCR7-CD45RA+CCR7-.
In HIV patients, HIV-specific CD8+ T cell
populations appear to be composed predominantly of the preterminally
differentiated
CD45RA-CCR7- subset,
while, at the same time, CMV-specific CD8+ T
cells display in large majority a terminally differentiated phenotype
(CD45RA+CCR7-). Thus,
it appears from the literature that T cells differentiate upon Ag
exposure through a continuum display of evolving functions that might,
in turn, affect the overall effectiveness of a given immune
response.
Epitope-specific vaccination offers the unique opportunity of evaluating the immune response in relation to a well-defined time of Ag exposure. In particular, immunization with the HLA-A*0201-associated gp100:209–217(210M) epitope (g209-2M) dramatically converts a largely undetectable T cell response in vaccine-naive patients to the frequently observable induction of vaccine-specific CD8+ T cells documentable by tHLA phenotyping (2). Thus, subsets of Ag-experienced/memory T cells can be characterized in their phenotypic and functional properties. Therefore, in this study we determined the expression of CD45RA and CD27 in epitope-specific CD8+ T cells. Expression was determined from the PBMC of patients undergoing vaccination with g209-2M modified from the wild-type epitope of the melanoma Ag gp100/Pmel17 by substitution of a methionine in position 2 (16). This peptide has been previously shown to efficiently induce epitope-specific CD8+ T cell responses in most patients when administered emulsified in IFA (17). These responses could be easily identified using tHLA complexes reconstituted with either the g209-2M or the wild-type gp100:209–217 (g209) peptide (2). Thus, the goal of this study was to characterize the CD45RA CD27 profile of tHLA+ vaccine-induced CD8+ T cells with the assumption that these cells are Ag-experienced, because none was detectable before vaccination. CCR7 expression was also evaluated in a subpopulation of patients because of its predictive significance concerning the ability of T cells to localize and exert effector function in the target organ. In addition, perforin expression was included in the phenotypic characterization as a marker of "ready for action" effector activity. More specifically, in this study we questioned whether these molecules could be regarded as informative markers of the level of differentiation and/or function of immunization-induced T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study we randomly selected 13 patients with metastatic melanoma among 20 who demonstrated a specific immune response to vaccination with a gp100 peptide. These 20 immunization-responsive patients were identified among 35 HLA-A*0201 patients with melanoma. These 35 patients had received repeated s.c. injections of the g209-2M peptide in IFA in a protocol approved by the institutional review board of the National Cancer Institute. No concomitant treatment, including IL-2, was administered. The HLA class I phenotype of patients was determined in PBMC using sequence-specific primer-PCR (18). All patients had been surgically determined to be without evidence of disease before enrollment in the vaccination protocol that was administered with adjuvant purposes. Thus, no correlation with clinical outcome could be evaluated in this study.
Peptide
The g209-2M (IMDQVPFSV) peptide used for vaccination was prepared according to good manufacturing practice by Multiple Peptide Systems (San Diego, CA). The g209-2M peptide used for tHLA synthesis and IVS studies was commercially synthesized by Princeton Biomolecules (Columbus, OH). The peptides were purified by gel filtration to >95% purity, and their identities were confirmed by mass spectral analysis.
Cells and culture conditions
PBMC were obtained by leukapheresis both before the patients received their first vaccine and 3 wk after vaccination. As discussed in Results, samples were obtained from patients who had received different numbers of vaccinations administered according to various time schedules. PBMC were isolated by Ficoll gradient separation and frozen until analysis. Analysis of vaccine-specific T cells was performed after overnight resting of thawed PBMC in complete medium consisting of Iscove’s 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), and 10% heat-inactivated human AB serum (Gemini Bioproducts, Calabasas, CA). This step allowed depletion of adherent monocytes.
The PBMC were also analyzed 10 days following IVS with 1 µM g209-2M peptide, followed by culture in IL-2 (300 IU/ml)-containing medium that was added on the day following stimulation and every 2–3 days thereafter. Bulk CTL cultures were cloned by limiting dilution according to a modification of Riddell’s technique (19, 20). Briefly, bulk CTL cultures were plated in 96-well plates at 0.6 cell/well with OKT3 (30 ng/ml), 50,000/well irradiated allogeneic PBMC (3,000 rad), and IL-2 (300 IU/ml). The clone used for this study (P1G9) was selected according to its reactivity against HLA-matched, gp100-expressing tumor targets as previously described (21). For stimulation of T cells, two melanoma cell lines were used characterized by expression (624.38 MEL) or lack of expression (624.28 MEL) of HLA-A*0201.
Kinetics of surface marker expression in response to Ag-specific stimulation
The g209- and g209-2 M-specific CTL clone P1G9 was cocultured in the presence of the HLA-A*0201/gp100-expressing melanoma cell line 624.38 MEL or a sister clone (624.28 MEL) that lacked expression of HLA-A*0201 but retained expression of gp100 (22, 23). Coculture was performed at a 1:4 cancer cell:CTL ratio in a six-well Costar plate (Corning Glass, Corning, NY) in complete medium at a concentration of 106 CTL/ml. Twenty-four hours after starting the coculture IL-2 (300 IU/ml) was added and was replenished every 2–3 days. Cocultured cells were harvested at various time intervals (as described for individual experiments) and analyzed by FACS analysis. To simulate the conditions of high epitope density used for IVS of PBMC, P1G9 clone was also stimulated in vitro with 1 µM g209-2M peptide. However, this stimulation could not be performed by directly adding soluble peptide to the medium as was done for the PBMC because this caused extensive apoptotic death of the T cell clone. To avoid this, g209-2M peptide was pulsed on 624.38 MEL and 624.28 MEL for 1 h and then washed away. The melanoma cell lines were then used for epitope presentation in conditions identical with those described previously.
T cell staining
Tetrameric peptide-HLA-A*0201 complexes were produced as
described previously (2, 24). Recombinant HLA-A*0201 H
chain containing a biotinylation site and recombinant
2-microglobulin were synthesized and used for
refolding of soluble HLA molecules in the presence of g209-2M.
Monomeric HLA/peptide complexes were biotinylated with BirA (Avidity,
Denver, CO) and tetramerized by adding avidin-PE (Pierce,
Rockford, IL).
Purified anti-CD27 mAb was obtained from BD PharMingen (San Diego, CA) and was used with the secondary PE-conjugated rat anti-mouse IgG1 from BD Biosciences (San Jose, CA). PE-conjugated anti-CD27 mAb was obtained from BD PharMingen. FITC-conjugated mAb against CD45RA was obtained from Caltag Laboratories (Burlingame, CA). Purified CCR7-specific mAb was obtained from BD PharMingen and was used with the secondary CY5.5-conjugated rat anti-mouse IgM from BD Biosciences. Perforin staining was performed using the BD Biosciences kit. CD8 mAb was obtained from BD Biosciences.
After overnight depletion of monocytes (or 10–11 days following IVS),
nonadherent PBMC were resuspended at 106 cells/50
µl FACS buffer (phosphate buffer plus 5% FCS; Biofluids). Cells were
incubated at 4°C with 1 µl tHLA for 15 min, and then incubation was
continued for 30 min with the specific mAb. A similar staining
procedure was applied for staining CTL cultures and the CTL clone P1G9.
After tHLA staining, cells were directly stained with Abs for surface
markers (CD27, CD8, CD45, CCR-7) and kept for 45 min at 4°C or fixed
in 4% paraformaldehyde and then permeabilized with acetone as
previously described (25) for perforin staining. The
totally CD8+ P1G9 clone was stained with CD27-PE,
CD45RA-FITC, tHLA-PE, CD8-PerCP, and V17-FITC (Endogen, Woburn, MA).
After staining, cells were washed in 2 ml FACS buffer and analyzed by
FACS (BD Biosciences). The acquisition data were analyzed after
pre-gating for lymphocyte size and tetramer positivity. The CTL clone
P1G9 was discriminated from cocultured cancer cells based on forward
scatter as described in Results. Twenty thousand events were
acquired for analysis of the T cell clone, and 200,000 were used for
PBMC or CTL after IVS.
Statistical analysis
Vaccine-specific Tc-pf was calculated as the number of g209 tHLA-staining CD8+ T cells per 106 CD8+ cells adopting the following formula: f = URQ/(URQ + LRQ) x 106 CD8+ cells, with URQ (upper right quadrant) containing the tHLA+CD8+ cells and LRQ (lower right quadrant) containing all other CD8+ cells. From these frequencies the background with CD8+ staining only was subtracted for each sample to obtain the corrected frequency. The corrected Tc-pf is presented as the number of vaccine-specific T cells per 106 CD8+ T cells.
Comparison of values between pre- and postimmunization specimens or comparing identical specimens before and after IVS was performed using a paired sample t test. The p values are reported as the level of significance for a two-tailed analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMC from 13 HLA-A*0201-expressing patients with metastatic
melanoma undergoing repeated immunization with s.c. injection of
g209-2M peptide emulsified in IFA were phenotypically characterized by
g209-2M tHLA staining of CD8+ T cells. For this
study PBMC were selected from patients who had developed detectable
vaccine-specific Tc-pf after immunization (Fig. 1
). Tc-pf was considered detectable when
>1,000 g209-2M tHLA-staining T cells were counted per
106 CD8+ T cells. This
value, according to our previous experience, corresponded to evidence
of immunization and correlated with results obtained by IVS
(2) or functional ex vivo assays (7). In none
of these patients could >1,000 g209-2M tHLA staining T
cells/106 CD8+ T cells be
detected in vaccine naive settings, a finding consistent with previous
reports (2, 4, 7). After immunization, Tc-pf ranged
between 1,600 and 46,100 x 106
CD8+ T cells. This broad range in Tc-pf is not
uncharacteristic of vaccine-induced immune responses. In this
particular case the different schedule of administration and the
different number of vaccinations received by individual patients (Table I) might have affected Tc-pf
(4). In fact, the patients included in this study were
among those participating in a randomization protocol in which
different schedules of administration of the g209-2M in IFA were
compared. According to the arm assigned, the patients received the
peptide injection weekly or at 3-wk intervals or for 4 consecutive days
at 3-wk intervals. In each arm of the study the patients received four
cycles of the assigned schedule and were then immunologically
reassessed 3 wk after administration of the last peptide injection by
obtaining a leukapheresis to compare vaccine-induced Tc-pf with
pretreatment samples. This study was designed to compare the
effectiveness of different administration schedules, and it is still
under investigation. Therefore, the results are beyond the purpose of
this report. However, PBMC were randomly sampled from individuals who
were treated in different arms of this protocol and who had received a
different number of vaccinations to address whether any of these
factors may bear any effect on the phenotypic characteristics of
vaccine-induced T cells and deserve further investigation. Thus, the
only constant parameter studied here is the phenotypic and functional
portrait of vaccine-induced T cells obtained after different
vaccination schedules 3 wk after the last administration of vaccine.
Results obtained from the 13 patients studied suggested, as shown in
the next section, that this factor significantly affected the CD45RA
CD27 CCR7 phenotypic profile of vaccine-induced T cells and, therefore,
the diversity of treatment schedule and the number of vaccinations were
not investigated further. Thus, in all patients selected for this study
Ag-experienced circulating CD8+ T cells were
detected 3 wk after the last immunization that could be further
characterized (9).
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Vaccine-induced
tHLA+CD8+ T cells were
further characterized according to the classification suggested by
Hamann et al. (9) based on CD45RA and CD27 expression
(Fig. 2). The following subtypes were
considered: CD45RA+CD27+
(naive), CD45RA-CD27+
(memory), CD45RA+CD27-
(effector), and
CD45RA-CD27-
(effector/memory) (Table II). Circulating
vaccine-induced T cells analyzed ex vivo were characterized by a small
size, as demonstrated by side and forward scatter, suggesting a small
size characteristic of resting T cells. In this respect they did not
display any morphological feature significantly different from the
remaining circulating CD8+ T cells. In the
majority of cases vaccine-induced T cells belonged to effector (43
± 6% of tHLA-staining CD8+ T cells) and
effector/memory (30 ± 4.1%) subsets, while only a small
proportion displayed naive or memory markers. Because Hamann et al.
(9) had previously reported that memory type
CD8+ T cells are characterized by a high
expression of perforin, we tested the expression of this cytotoxin in
tHLA+CD8+ T cells.
Surprisingly, only a small proportion of these cells expressed
detectable amounts of perforin (17 ± 4.5%). In addition, to
further characterize vaccine-induced T cells, we tested PBMC from six
patients for CCR7 expression that differentiates between central memory
and effector memory T cells characterized, respectively, by the
presence or the absence of expression of this marker (14).
Sixty-three percent of vaccine-induced T cells did not express CCR7. Of
them the majority (73%; 46% of all vaccine-induced T cells) were
CD27-, while a second smaller subpopulation was
CCR7+CD27+ (Table III). This observation suggested a
spectrum of vaccine-induced T cells of which the preponderance appeared
to bear two phenotypic characteristics associated with effector
(CD27-CCR7-) function.
Thus, vaccine-induced T cells appear to belong in large majority to an
effector phenotype. However, contrary to other reports these effector
cells appear deprived of perforin.
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CTL cultures from postvaccination PBMCs were expanded in 300 IU/ml
IL-2 for 10 days after IVS with 1 µM g209-2M. Forward and side
scatter analysis demonstrated that CD8+ T cells
(whether vaccine-specific or not) were condensed in a population of T
cells of a larger size than that observed in the generating PBMC (inset
in scatter plot, Fig. 2
). This increase in size was seen in all
cultures tested (P3–P13) and was consistent with the typical
morphology of IL-2 and/or stimulation of activated T cells after in
vitro culture. Phenotyping of these cultures demonstrated a significant
increase in CD27+ cells independent of the
expression of CD45RA (from 28 to 67%; by paired t test,
p = 0.01; Table II). In particular, 23% of IVS-derived
CTL were CD45RA+CD27+,
corresponding to a naive phenotype. However, their naive status was
considered unlikely, first because no evidence of tHLA staining T cells
could be identified before vaccination, and second because, as we have
repeatedly shown, IVS does not induce g209-2M CTL in vaccination-naive
circulating cells (2, 4). This suggests that it is
unlikely that the CD8+ tHLA-staining T cells
expressing both CD45RA and CD27 resulted from the in vitro expansion of
a subliminal number of Ag-inexperienced T cells undetectable by tHLA,
because this does not occur in PBMC obtained before vaccination. In
addition, CD45 RA positivity significantly decreased in response to IVS
from 54 to 32% of vaccine-specific T cells (P3–P13; by paired
t test p = 0.02). Interestingly, together
with changes in CD27 status, a dramatic increase in perforin expression
was noted among the tHLA-staining postvaccination CTL cultures (from
17 ± 4.5% to 84 ± 3.6%; by paired t test for
P3–P13, p < 0.001). Similarly, an almost complete
loss of expression of CCR7 was noted, with 93% of vaccine-induced T
cells being CCR7 negative (Table III). Taken together, these data
suggest that upon IVS the vaccine-elicited T cells undergo a further
step in the process of activation/differentiation that appears to
increase their effector potential, which includes larger cellular size,
decreased expression of CCR7, and increased expression of perforin. In
contrast, small cellular size and lack of perforin expression in
circulating PBMC in the absence of IVS and expansion suggest a resting
status of vaccine-induced CD8+ T cells. Puzzling,
however, remained the interpretation of the increased frequency of
CD27+ vaccine-specific T cells after IVS. It is
not clear whether the phenotypic changes observed after IVS were
due to the epitope-specific stimulation rather than to the presence of
IL-2 (300 IU/ml) in the culture medium. Analysis of tHLA-negative cells
after IVS (presumably nonstimulated by the epitope and, therefore,
responding predominantly to IL-2 stimulation) did not show the changes
noted in tHLA+ cells (data not shown). In
addition, in vitro culture of PBMC obtained from three
non-tumor-bearing healthy donors in the presence or the absence of IL-2
(300 IU/ml) and no epitope-specific stimulation did not induce a
preferential shift of CD8+ T cell phenotype.
Although some variation in the CD8+ T cell
phenotype was noted among different donors, no significant changes were
noted in the percentage of CD8+ T cells
expressing perforins after 10 days of in vitro culture compared with
fresh PBMC (Table IV). Interestingly, in
vitro culture of PBMC without IL-2 caused a marked decrease in the
percentage of perforin-expressing CD8+ T cells,
suggesting that IL-2 may play a role in sustaining, rather than
inducing, the effector function of CTL. The percentage of CD27,
CD45RA-expressing cells was not significantly affected by in vitro
culture with IL-2, nor was the expression of CCR7 (Table V).
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To test whether changes in CD27 expression define the status of
activation of vaccine-induced T cells rather than representing a marker
of their status of differentiation (naive vs memory vs effector), we
stimulated in vitro the g209-2M-specific CTL clone (P1G9) derived from
circulating lymphocytes of a melanoma patient who had received
immunization with the same epitope (4). This clone is
characterized by high affinity for the HLA/g209-2M complex, has a
classical effector phenotype
CD45RA+CD27-, and
expresses detectable amounts of perforin when cultured in vitro in the
presence of 300 IU/ml IL-2 (9). P1G9 was cocultured at a
1/4 ratio with either the HLA-A*0201-negative 624.28 or the
HLA-A*0201-expressing 624.38 melanoma cell line. IL-2 (300 IU/ml) was
added the following day and every 2–3 days thereafter. Before
stimulation and 24, 48, and 96 h after stimulation, tHLA staining
and V-17, CD8, perforin, and CD27 expression were analyzed by FACS
analysis. T cells were discriminated from cancer cells by forward and
side scatter, and the accuracy of this strategy was confirmed by DNA
index analysis as previously described (26) (Fig. 3A).
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17, perforin, and CD27 were noted as expected due to the lack of
TCR engagement with the melanoma cell line (Fig. 3B).
Stimulation with the HLA-A*0201-expressing sister clone 624-38
demonstrated a decrease in tHLA staining associated with
TCR/HLA-A*0201/epitope engagement as previously described
(7) (Fig. 3C). This decrease was associated
with no or minimal changes in the expression of other surface markers,
with a slight decrease in V-17 and CD8 expression. Thus, stimulation
of a T cell effector clone using HLA-A*0201-matched cell lines induced
minimal effects on the status of activation of such a clone, although
these conditions are sufficient to induce IFN- mRNA and protein
expression as well as reduction in tHLA staining as previously
described (7). Kinetics of stimulation of the effector-type g209-2M-specific CTL clone P1G9 with the melanoma cell line 624.38 (HLA-A2+gp100+) exogenously pulsed with 1 µM g209-2M peptide
The conditions used for IVS of PBMC differed from those used to
stimulate P1G9 with HLA-matched tumor cells, in that PBMC were
vigorously challenged with high density epitope exposure achieved by
exogenous administration of 1 µM peptide. We have used this technique
for quite a long time with the assumption that monocytic cells present
in the PBMC preparation would contribute to Ag presentation (2, 27, 28, 29). Using this technique we have previously noted that
decrements in the concentration of epitope used for cognate stimulation
tightly correspond with decreasing levels of IFN- mRNA expression by
PBMC, while down-regulation of tHLA staining is only minimally affected
(4). In addition, we have previously shown that variations
in epitope density on the surface of tumor cells strongly affects the
intensity of T cell stimulation (22, 30), which can be
greatly enhanced by incubation of cancer cells with soluble epitope
before coculture (30). Indeed, analysis of a panel of CTL
clones expanded from PBMC obtained from melanoma patients after
immunization showed a strong correlation between the level of
stimulation with tumor cells at a peptide concentration corresponding
to 0.03 µM (31). Thus, we questioned whether stimulation
with tumor cells expressing HLA-A*0201 and gp100 could be
quantitatively comparable to the high density peptide exposure
achievable with the saturating concentrations of g209-2M peptide (1
µM) used for PBMC stimulation (31).
To enhance the effects of the stimulation induced by the melanoma cell line 624.38-MEL, we therefore artificially increased its epitope density by loading tumor cells with 1 µM g209-2M peptide. Direct addition of g209-2M to the CTL clone was not feasible; preliminary experiments showed massive apoptosis of T cells (data not shown), possibly due to cross-recognition of HLA-epitope complexes on the surface of T cells or to Fas/Fas ligand interactions among activated T cells (32).
Upon exposure to g209-2M-loaded 624.38 melanoma cells, P1G9
demonstrated an immediate and profound down-regulation of the
expression of CD8 and the V-17 TCR chain expressed by this clone.
The down-regulation of CD8 and the clonotypic V- chain was
associated with loss of g209-2M tHLA staining (Fig. 4). In addition, exposure to cognate
stimulation decreased the expression of perforin within the first
24 h. In contrast, CD27 expression was up-regulated by 24 h
after stimulation and persisted for several days following stimulation.
During a period of 10 days in culture after epitope-specific
stimulation in the presence of 300 IU/ml IL-2, the expression of CD8
slowly reconstituted. This reconstitution of expression was associated
with restoration of V-17 expression, followed by reappearance of
tHLA staining. During the same period, perforin levels were
progressively restored, while the expression of CD27 remained
detectable. Thus, the P1G9 phenotype had switched from a canonical
effector type
(CD45RA+CD27-perforin+)
to a new hybrid phenotype CD45RA+,
CD27+, and perforin+
reminiscent of a canonical naive T cell
(CD45RA+CD27+).
Interestingly, at a particular time point (24 h after stimulation),
P1G9 had a classical naive phenotype: CD45RA+,
CD27+, and perforin-.
Because it is unlikely that this CTL clone had regressed to an
Ag-inexperienced status of differentiation, it is most likely that
switches in CD27 expression and perforin detectability reflect
different levels of activation of circulating and in vitro cultured T
cells. In this study we did not address in depth the differential role
that cognate stimulation vs IL-2 exposure may play in affecting changes
in T cell phenotype. However, we believe that the intensity of cognate
stimulation may have a predominant role. In one experiment in which the
same stimulatory conditions were applied to P1G9 (stimulation with
624.28 MEL or 624.38 MEL with or without exogenous loading with
g209-2M) in the presence or the absence of IL-2, no demonstrable
differences in expression of the previously discussed markers were
noted up to 4 days following stimulation (data not shown). It should be
emphasized that these data derived from a clonal population may have
limited relevance to the in vivo situation, as various CTL clones may
be quite heterogeneous depending on various intrinsic and extrinsic
factors. However, the switch in phenotype observed in this clone in
response to cognate stimulation suggests that markers such as CD27 may
vary independently of the status of differentiation of individual T
cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several factors may be responsible for the lack of correlation between
the identification of an immune response to the vaccine and the lack of
cancer regression. Obviously, circulating T cells induced by
vaccination may not reach the tumor site to exert their effector
function. However, attempts to expand vaccine-specific T cells from
biopsies through fine needle aspirates of the same lesion obtained
before and after treatment were more frequently successful in
postvaccination samples (42). In addition, we noted
accumulation of gp100-specific lymphocytes in metastatic deposits
following vaccination in 8 of 11 patients. This accumulation was
associated with increased expression of IFN- in lesions that
expressed gp100. Yet this localization was not sufficient for tumor
regression despite the expression by melanoma cells of the gp100 Ag
targeted by the vaccine (43). These findings suggest that
vaccine-elicited T cells can localize within the target tissue and that
they are engaged by tumor cells to produce IFN-, but this is not
sufficient, and additional factors may be responsible for their lack of
effect on tumor growth.
It could also be postulated that vaccine-induced Tc-pf, although clearly observable, may not be sufficient to induce tumor regression, because the number of immunization-induced T cells observed in this study may not be above the threshold that may be required to eliminate all tumor cells. In a mouse model we identified a correlation between the intensity of immune response induced by the immunogen and tumor rejection. This correlation could be easily identified in this experimental model, because the use of a clonal population of tumor cells and of inbred animals reduced tumor heterogeneity and/or variability of individual animal immune responses providing a more homogeneous target for immunotherapy (44). However, in humans a clear quantitative correlation between the extent of immune response and clinical outcome has remained elusive in most reported clinical trials, perhaps in relation to the heterogeneity of tumors in various individuals affecting their immune responsiveness (45).
T cell responses documented by IVS or by direct ex vivo enumeration of
Tc-pf with vaccine-specific tHLA may not fully address their status of
activation in vivo. In fact, Lee et al. (5) have shown
that tumor-specific T cells identified in vaccine-naive patients by
tHLA may not be able to exert effector function either through target
killing or expression of cytokines such as IFN-. We have previously
argued that this concept may not pertain to vaccine-induced
CD8+ T cell responses, because in at least a
percentage of them, it is possible to document the expression of
IFN- upon cognate stimulation with vaccine-specific epitope or
HLA-matched tumor cells (4, 5, 6, 7). However, recent reports
have emphasized a dichotomy between IFN- secretion by T cells and
their killer function (8). This suggests that IFN-
expression in itself may not be the ultimate arbiter of T cell effector
function, but may represent a sensitive marker of T cell reactivity to
cognate stimulation.
Hamann et al. (9) suggested a spectrum of circulating T cell phenotypes from naive to effector types based on the expression of several cellular markers of which CD45RA and CD27 appeared to best predict their status of differentiation and consequently their function. Others suggested that homing receptors might be responsible for the effector function of T cells by regulating their localization within target organs (14). The observation in this and other studies (6) that vaccine-elicited T cells express low or undetectable CCR7 suggests that homing may not be, at least based on this marker, a significant problem in the context of vaccines. This finding is consistent with an effector phenotype reported for flu- and CMV-specific T cells (6, 15) and correlates with our observation that vaccine-induced T cell responses can be detected in lesions resistant to therapy following vaccination (42, 43).
Segregation of T cell subsets into naive, memory, and effector types suggests a static and irrevocable status of differentiation that may not necessarily apply to the dynamic behavior of surface markers in response to T cell stimulation. For instance, expression of CD27 may play a role in the survival of activated T cells (46). Stimulation of the TCR complex with PHA or CD3 mAbs causes a dramatic increase in CD27, and addition of CD27 Abs to T cell cultures leads to enhanced proliferation (47). Interestingly, CD27 could be immunoprecipitated from the membrane of activated, but not resting, T cells (47). Recently, CD27 was described as a costimulatory receptor responsible for the generation and maintenance of T cell memory responses (48). Our findings suggest that CD27 expression in circulating vaccine-induced T cells may represent a marker of their status of activation rather than differentiation. Indeed, CD27 could be induced through IVS of vaccine-induced CD27- (effector) CD8+ T cells. This increase in CD27-expressing T cells after 10 days of IVS is probably the result of its de novo expression by CD27- PBMC, rather than the preferential expansion of few CD27+ T cells. In particular, the substantial number of CD45RA+CD27+ (naive) CD8+ T cells (23%) observed after IVS is unlikely to be the result of expansion of rare (below the threshold of detection of tHLA) Ag-inexperienced precursors in PBMC obtained after vaccination. We believe that all the vaccine-specific T cells identifiable through IVS after immunization must have been exposed to the vaccine epitope, because we could never expand g209-2M-specific T cells before vaccination using the exact in vitro conditions used here (2, 17). Thus, postvaccination IVS-induced T cells are most likely Ag-experienced derived from the CD45RA+CD27- PBMC pool. The CD45RA expression in Ag-experienced T cells is reminiscent of the stable CD45RA+LFAhigh memory state recently described by Faint et al. (49). Unfortunately, because of the limited number of cells available, LFA-1 as well as other potentially interesting markers such as CCR5, CXCR1, and CD62 could not be evaluated in this study. The demonstration that a vaccine-induced, effector-type T cell clone could reverse its phenotype from CD27- to CD27+ upon IVS suggests that the phenotype switch observed in IVS-expanded CTL is due to a change in functional status rather than preferential expansion of T cells in a particular status of differentiation. Thus, as suggested by others (6), CD27 may not be the most accurate surface marker to segregate naive from Ag-experienced CD8+ T lymphocytes as also reported for CD45RA (49). Indeed, expression of the CD45RA isoform has been noted to occur during the quiescent stage of memory T cell reversion to a resting state after in vivo priming by acute EBV infection (49). Interestingly, the phenotypic changes observed on P1G9 could only be induced by intense stimulation with saturating conditions of epitope (pulsed tumor cells), while exposure to tumor cells expressing natural amounts of HLA/epitope on their surface was not sufficient to induce such changes even in the presence of exogenous IL-2. This finding suggests that in vivo contact between vaccine-induced T cells and tumor cells may not be sufficient to induce productive T cell stimulation capable of sustaining a brisk immune response as suggested by others (50, 51).
Of particular significance in this study was the lack of expression of perforin in circulating vaccine-induced T cells. This in association with their small size and their lack of expression of CD27 suggests that vaccine-induced circulating T cells are in a resting status, which can be reversed by IVS. It was beyond the purpose of this study to address in depth whether this in vitro reactivation is due to re-exposure to cognate stimulation or to the 10-day culture with IL-2. Whatever the cause of the striking difference noted after 10 days of IVS, this change in phenotype may suggest a reason for the discrepancy between satisfactory function of T cells observed in vitro after IVS and their limited function in vivo. This study also did not address when and where vaccine-induced T cells turn into this postulated resting phase. All samples in this study were obtained 3 wk after immunization. Thus, it is possible that this period marks the gradual regression of vaccine-induced T cells toward a resting/memory phenotype. Analysis of vaccine-induced Tc-pf at an earlier time point might have depicted a totally different portrait. Perhaps a more intense and consistent exposure in vivo to vaccine may induce and sustain a more active and functionally rewarding immune response as suggested by others (50, 52, 53).
In summary, we hypothesize that vaccine-induced T cells belong to an
effector subtype (predominantly CD27- and
CCR7-) that is in a resting phase characterized
by lack of perforin, small cellular size, and lack of expression of
activation markers such as CD27. These cells, however, are not totally
anergic and, on the contrary, are responsive to cognate stimulation, as
they can produce IFN- upon exposure to relevant Ag (4, 7). It is possible that if the stimulus is delivered in optimal
conditions their full effector function can be restored as modeled by
the 10-day IVS conditions. These findings assign a limited level of
functionality to circulating vaccine-induced T cells and may partially
explain why increases in Tc-pf following immunization may not directly
correlate with clinical cancer regression.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Francesco M. Marincola, Immunogenetics Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Building 10, Room 1C-711, 10 Center Drive, MSC 1502, Bethesda, MD 20892-1502. E-mail address: fmarincola{at}mail.cc.nih.gov
3 Abbreviations used in this paper: Tc-pf, T cell precursor frequency; g209, gp100:209–217; g209-2M, g209(210 M); IVS, in vitro stimulation; tHLA, HLA/epitope tetrameric complex.
Received for publication January 4, 2002. Accepted for publication March 27, 2002.
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) and after (
) vaccination in 13 patients
undergoing immunization with g209-2M peptide administered s.c.
emulsified in IFA. Tc-pf is shown as the number of tHLA-staining T
cells per 106 CD8+ T cells.
