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*
Surgery Branch, Division of Clinical Sciences, National Cancer Institute,
Department of Transfusion Medicine, Clinical Center, 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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expression detectable by IC-FACS or qRT-PCR,
respectively, in five and six of these patients. Furthermore,
down-regulation of tHLA staining was noted upon cognate stimulation
that could be utilized as an additional marker of T cell
responsiveness. Finally, we observed in six patients an enhancement of
reactivity against vaccine-matched tumor targets that was partly
independent of documented vaccine-specific immune responses. A strong
correlation was noted between tHLA staining of postvaccination PBMC and
IFN- expression by the same samples upon vaccine-relevant
stimulation and assessed either by IC-FACS or qRT-PCR. Thus, blunting
of the status of T cell activation on itself cannot easily explain the
lack of clinical responses observed with
vaccination. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the past, we have judged the extent of the immune response to
epitope-specific vaccination by comparative assessment of the
sensitivity of pre- and postvaccination PBMC to in vitro stimulation
with arbitrary concentrations of vaccine-related epitopes plus
exogenous IL-2 (1). This approach may misrepresent and
likely exaggerate the extent and quality of vaccine-induced responses
occurring in vivo. Recently, direct measurement of circulating
vaccine-specific T cells precursor frequency (TCPF) with HLA/epitope
tetramers (tHLA) demonstrated a tight correlation between their
frequency and susceptibility to in vitro expansion (8).
This correlation suggested that historical results obtained with in
vitro sensitization were roughly representative of the host’s immune
competence before and after vaccination. However, tHLA-guided detection
of vaccine-specific T cells does not address their functional status
(7). Indeed, tHLA binding does not exclude that a
promiscuous, low avidity TCR/tHLA interaction has occurred that cannot
generate ligand-induced physiological responses (7, 9). In
addition, even a high avidity TCR may not generate productive
interactions with its natural ligand due to a diminished status of
activation of the T cell bearing it (7). In a previous
study, serial measurement of cytokine gene expression by quantitative
real-time PCR (qRT-PCR) detected functional immune responses to
vaccination in circulating lymphocytes and at tumor site
(10). However, because qRT-PCR does not identify the cells
expressing the transcript of interest, such responses could not be
directly attributed to tHLA-determined, CD8+ T
cells elicited by the vaccine (7). Therefore, the purpose
of this study was to combine methods that directly enumerate and
characterize the in vivo status of activation of vaccine-elicited T
cells with minimal ex vivo manipulation. The immune response to
vaccination of eight patients with metastatic melanoma who received
repeated immunization with gp100:209–217(210 M) (g209-2 M) peptide in
IFA was monitored using tHLA, qRT-PCR, and intracellular measurement of
IFN- production by FACS analysis (IC-FACS) (11). A
strong correlation was noted between the phenotypic identification by
tHLA of vaccine-specific T cells and expression of IFN- in response
to relevant stimulation, suggesting that blunting of the status of
activation of circulating T cells cannot be easily shown to be a
significant factor determining failure of vaccination.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight HLA-A*0201-expressing patients with metastatic melanoma (P1 to P8) received repeated s.c. injections of the gp100:209–217(210 M) (g209-2 M) peptide in IFA. Vaccinations were administered either at 1-wk (P6, P7) or 3-wk (P1, P2, P3, P4, P5, P8) intervals, and blood samples for PBMC extraction were obtained just before the next vaccination and 3 wk after the prior vaccination. The HLA class I phenotype of patients was determined on PBMC using sequence-specific primer PCR (12). PCR was also used for molecular subtyping of HLA-A*02 (13).
Cells and cultures
Samples were obtained from leukaphereses of patients before and after vaccination with peptides. PBMC were isolated by Ficoll gradient separation and frozen until analysis. 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 (Cetus Oncology Division, Chiron, Emeryville, CA). This step allowed depletion of adherent monocytes. The melanoma cell clones 624.38 MEL and 624.28 MEL cells were established and cultured as previously described (14). These clones were derived from the same bulk culture and are identical in expression of MA and HLA alleles with the exception of HLA-A*0201, whose expression is lost in 624.28-MEL due to aberrant splicing of HLA-A*0201 heavy chain mRNA (15). The breast cancer MCF-7 and the lymphoblastoid T2 cell lines were purchased from the American Type Culture Collection (Manassas, VA). The g209/209-2 M-reacting T cell clone C1–35 and 1520 H-3.1 have been previously described (16), while the T cell clones C6, H5, B3, and C11 were expanded and isolated according to a previously described protocol (16, 17) from bulk cultures of earlier patients’ PBMC who had received g209-2 M vaccine.
Peptides
The peptides used for vaccination were prepared according to Good Manufacturing Practice by Multiple Peptide Systems (San Diego, CA). The identity of each of the peptides was confirmed by mass spectral analysis. Peptide sequences are described below as relevant. The same peptides were used for stimulation of T cells.
Epitope-specific T cell staining using HLA-A2 tetramer
Tetrameric peptide-HLA-A*0201 complexes were produced as described previously (8, 18). Recombinant HLA-A*0201 heavy chain containing a biotinylation site and recombinant ß2-microglobulin were synthesized and used for refolding of soluble HLA molecules in the presence of a HLA-A*0201-binding peptide. Soluble HLA molecules were prepared for the following epitopes: gp100:209–217 (210 M) (IMDQVPFSV, g209-2 M); MART-1:26–35(27L) (ELAGIGILTV, MART-2L); and Flu-M1:58–66 (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 soluble HLA on gel filtration. Monomeric HLA/peptide complexes were biotinylated with BirA (Avidity, Denver, CO) at the heavy chain and separated from free biotin by gel filtration. Biotinylated HLA complexes were tetramerized by adding avidin-PE (Pierce, Rockford, IL) at a 4:1 molar ratio. The final concentration of tetramer was adjusted to 2 mg/ml. 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). Cells were incubated on ice with 1 µg tHLA for 15 min, then cultured 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). Ten thousand events were acquired for analysis of T cell clones and 200,000 for PBMC samples.
FACS analysis for intracellular expression of IFN-
Nonadherent PBMC (1 x 106 cells)
were stimulated for 6 h by directly adding soluble peptide (1
µg/ml). After 2 h, Brefeldin A (10 µg/ml; Sigma, Deisenhofen,
Germany) was added. After additional 4 h, the cells were treated
with 4500 U DNase I (Calbiochem, San Diego, 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/PBS-S. Cells were permabilized and blocked
overnight with PBS/Saponin/5% milk at 4°C. After staining with mAb
for 30 min on ice, cells were washed in PBS-S. Staining with tHLA was
performed before fixation of cells. All samples were analyzed on a
Becton Dickinson FACSCalibur flow cytometer using the CellQuest
software. The following mAb were used: allophyocyanin-conjugated mouse
anti-human CD3 (IgG1), peridinin chlorophyl protein-conjugated
mouse anti-human CD8 (IgG1), and FITC-conjugated mouse
anti-human CD45RA were purchased from Becton Dickinson (Heidelberg,
Germany); FITC-conjugated mouse anti-human IFN- (IgG1) and
FITC-conjugated mouse anti-human CD45RO were purchased from
PharMingen.
Direct molecular assessment of peptide and melanoma-specific CTL reactivity
Cryopreserved PBMC were thawed into complete medium, as described (2). Direct PBMC assays were conducted using 3 x 106 PBMC in 2 ml of media. Thawed PBMC were allowed to recover by incubation at 37°C in 5% CO2 for 10 h. A total of 1 µM of peptide or 1 x 106 melanoma cells was then added to the PBMC and incubated at 37°C in 5% CO2 for 2 h. Soluble peptide was added rather than peptide pulsed onto T2 or other APCs, because these cells have high expression of costimulatory and adhesion molecules that could have exaggerated the responsiveness of the PBMC. No exogenous cytokines or other stimulants were added. The cells were then harvested for RNA isolation and cDNA transcription.
Quantitative real-time PCR
RNA isolation from PBMC was performed in batches containing
patient pre- and posttherapy samples with RNeasy mini kits (Qiagen,
Santa Clarita, CA). The RNA was eluted with water and stored at
-70°C. For cDNA synthesis, about 1 µg of total RNA was transcribed
with cDNA transcription reagents (Perkin-Elmer, Foster City, CA) using
random hexamers and directly tested. Measurement of gene expression was
performed utilizing the ABI prism 7700 Sequence Detection System
(Perkin-Elmer), as previously described (19, 20). Primers
and Taq Man probes (Custom Oligonucleotide Factory, Foster
City, CA) were designed to span exon-intron junctions to prevent
amplification of genomic DNA and to result in amplicons <150 bp to
enhance efficiency of PCR amplification (10).
Taq Man probes were labeled at the 5' end with the reporter
dye molecule FAM (6-carboxy-fluorescein; emission
max = 518 nm) and at the 3' end with the
quencher dye molecule TAMARA (6-carboxytetramethyl-rhodamine; emission
max = 582 nm). cDNA standards were generated
by reverse-transcriptase, primer-specific amplification of mRNA of the
relevant genes using a technique identical to the one used for the
preparation of test cDNA. Amplified cDNA was then purified and measured
by spectrophotometry (A260 OD). Copies were calculated using the m.w.
of each individual gene amplicon. RT-PCR of cDNA specimens and cDNA
standards were conducted in a total volume of 25 µl with 1x
Taq Man Master Mix (Perkin-Elmer). Thermal cycler parameters
included 2 min at 50°C, 10 min at 95°C, and 40 cycles involving
denaturation at 95°C for 15 s, annealing/extension at 60°C for
1 min. Real-time monitoring of fluorescent emission from cleavage of
sequence-specific probes by the nuclease activity of Taq
polymerase allowed definition of the threshold cycle during the
exponential phase of amplification (19). All PCR assays
were performed in duplicates and reported as the average. qRT-PCR was
performed for IFN- and IL-2 mRNA expression and normalized to copies
of CD8 mRNA from the same sample. Data were adjusted for CD8 mRNA
copies on the basic immunologic assumption that stimulation with an HLA
class I-restricted epitope defines CD8+ T cells
as the only relevant population. Since the frequency of
CD8+ T cells varies in time in individual
patients, it is incorrect to present data corrected by expression of
housekeeping genes, such as GAPDH or ß-actin, expressed by any cell
as done in other qRT-PCR applications (19, 20).
Statistical analysis
TCPF was calculated as previously described (8).
CD3+/CD8+ T cells were
gated, and the fluorescence intensity of tHLA and IFN- staining
reported in the y- and x-axis, respectively. The
frequency (f) of tHLA+ T cells
per 100 CD8+ T cells was calculated according to
the formula: ftHLA = upper
left quadrant (ULQ)/(ULQ + lower left quadrant (LLQ)) x 100 with the
upper left quadrant containing the tHLA+,
CD8+ T cells, and the left lower quadrant the
tHLA-, CD8+ T cells. From
these frequencies, the background positivity in samples prepared with
CD8 mAb but without tHLA was subtracted to correct for nonspecific
fluorescence in the ULQ. IFN--TCPF was calculated according to the
following formula:
fIFN- = upper
right quadrant (URQ) + lower right quadrant (LRQ)/(URQ + LRQ +
LLQ) x 100 with the right quadrants containing the
IFN-+, CD8+ T cells, and
the left quadrants the IFN--,
CD8+ T cells. From these frequencies, the
background was subtracted as for tHLA fluorescence.
The various methods were compared by adopting as independent parameter
the data accrued with tHLA staining of PBMC. Postvaccination samples
were divided in positive or negative for enrichment of tHLA staining
based on a 
3-fold increase in frequency of staining cells compared
with prevaccination samples. Positive and negative samples were then
examined for concordance with other methods by Spearman rank
correlation analysis. The following dependent parameters were
correlated with tHLA staining. 1) tHLA staining after stimulation of
PBMC with 209 and 209-2 M peptide. Positive samples were considered
those in which at least a 50% decrease in TCPF was noted compared with
unstimulated samples (None column in Table I
). 2) tHLA staining after stimulation of
PBMC with HLA-A*0201 expressing and not expressing melanoma cells
(Table II). Positive samples were
considered those in which the difference in TCPF between
postvaccination PBMC stimulated with HLA-A2+ or
HLA-A2- negative melanoma cells was at least
3-fold the difference in prevaccination PBMC. 3) IC-FACS staining for
IFN- in samples stimulated with 209 or 209-2 M peptide. Positive
samples were considered those with IFN- TCPF at least 3-fold above
pretreatment samples (Table I). 4) IC-FACS staining for IFN- in
samples stimulated with HLA-A*0201 expressing and not expressing
melanoma cells. Positive samples were considered postvaccination PBMC
in which the difference in frequency of stained cells stimulated with
HLA-A2+ and HLA-A-
melanoma was at least 3-fold the difference in prevaccination samples
(Table II). 5) qRT-PCR identification of vaccine-induced IFN-
expression in 209 and 209-2 M stimulated postvaccination PBMC
(Table I). Positive samples were postvaccination PBMC with IFN- mRNA
expression at least 3-fold above prevaccination samples. 6) qRT-PCR
identification of vaccine-induced IFN- expression in samples
stimulated with HLA-A*0201 expressing and not expressing melanoma
cells. Positive samples consisted of postvaccination PBMC, in which the
ratio of mRNA expression between samples stimulated with
HLA-A2+ and HLA-A2- cells
was at least 3-fold the difference in prevaccination samples (Table II). Statistical significance for each pair analyzed is presented as
Spearman’s 
correlation coefficient and respective p
value.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vaccine-specific T cells were enumerated by comparing g209-2
M/tHLA TCPF in pre- and postvaccination PBMC (Table I
). In six patients
(P3, P4, P5, P6, P7, and P8), a more than 3-fold enhancement of
vaccine-elicited TCPF was noted. In one additional patient (P2), a high
TCPF of vaccine-specific T cells was also noted. However, in this case,
the high TCPF could not be attributed to the vaccine because
prevaccination specimens convincingly demonstrated high frequency of
circulating T cells that stained with tHLA (Fig. 1). No constitutive expression of IFN-
was noted in any PBMC population independently of the detection of
vaccine-specific T cells by tHLA.
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tHLA-based TCPF were compared with the ability of PBMC to produce
IFN- upon stimulation with epitopes relevant to the vaccination such
as g209-2 M (vaccine) or the parental g209. This functional component
of the immune response to vaccination was addressed using IC-FACS
analysis and qRT-PCR (Table I). We, however, consistently noted that
stimulation caused a down-regulation of tHLA staining that accompanied
IFN- expression (Fig. 2). To better
characterize the down-regulation of tHLA staining in response to
stimulation, we examined the behavior of a clone (1520 H.3-1) from a
tumor-infiltrating lymphocyte (TIL) that naturally recognizes the g209
epitope and cross-reacts with g209-2 M. This
CD8+TIL was expanded from a melanoma metastasis
by exogenous administration of IL-2 and without ex vivo exposure to
Ag/epitope-specific stimulation (16). Down-regulation and
IFN- expression occurred upon exposure of 1520 H.3-1 to tHLA/209 (or
tHLA-209-2 M) at 37°C, but not 4°C, and correlated with CD69
up-regulation (Fig. 3). To avoid the
direct effect of tHLA on the intensity of staining of epitope-specific
T cells at 37°C, all stainings in this study were performed,
therefore, at 4°C. Interestingly, CD3 detection decreased in response
to incubation of 1520 H.3-1 with tHLA at 37°C; however, it was never
of the extent characterizing tHLA-staining down-regulation (data not
shown). This persistence of CD3 had the important practical advantage
of allowing sorting of CD3+ cells in the
four-color system utilized for FACS analysis in this study.
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(Fig. 4a). Stimulating
at 4°C reversed both phenomena (data not shown). Furthermore,
coculture with an HLA-A*0201/gp100 expressing melanoma cells (624.38
MEL), but not an HLA-A*0201-negative melanoma (624.28 MEL) nor a breast
cancer (MCF-7) cell line caused dramatic down-regulation of tHLA
staining and production of IFN- (Fig. 4b). Also in this
case, stimulation of 1520 H-3.1 at 4°C reversed down-regulation and
ability to produce IFN- (not shown). Thus, an inverse correlation
exists between tHLA staining of CD8+ T cells and
their activation that could be considered an additional, though
indirect, sign of productive TCR engagements (Table I). As a
consequence of staining down-regulation, however, stimulation concealed
vaccine-specific T cells, not allowing direct attribution of IFN-
expression to tHLA-stained T cells.
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in response to g209-2 M and g209 peptides
(P3, P4, P5, P6, and P8). Another patient (P7) demonstrated equivocal
results. In the remaining patients, no vaccine-responsive T cells could
be detected. Furthermore, PBMC from the patient with high TCPF in the
vaccine-naive status (P2) were unresponsive to vaccine-related stimuli.
Spearman rank correlation analysis demonstrated a strong correlation
between detection of vaccine-enhanced TCPF and IFN- expression by
IC-FACS ( = 0.78 for both g209 and g209-2 M-stimulated PBMC;
p = 0.005). Measurement of IFN- transcript by
qRT-PCR matched the results observed by IC-FACS and detected vaccine
responsiveness in one additional patient (P7). The discrepancy between
the results obtained by IC-FACS and qRT-PCR was consistent upon various
retesting and is not easily explainable, as higher sensitivity of
qRT-PCR over other methods as not been, to this point, demonstrated.
Also, in the case of qRT-PCR, a strong correlation was noted between
tHLA-based TCPF and IFN- expression (Spearman’s = 0.81 and
0.90 for g209 and g209-2 M-stimulated PBMC, respectively;
p = 0.003 and 0.001). Expression of IL-2 mRNA was also
tested by qRT-PCR. In five patients (P4, P5, P6, P7, and P8), it was
possible to identify a more than 3-fold increase in IL-2 transcript
compared with prevaccination PBMC (data not shown). This evaluation of
vaccine-specific T cells, therefore, suggested that repeated
administration of minimal epitope sequences in IFA could enhance the
frequency of functional CD8+ T cells in patients
with metastatic melanoma. Interestingly, vaccine-naive PBMC from P2
that demonstrated high tHLA-based TCPF did not demonstrate significant
levels of IFN- production upon stimulation, suggesting that
promiscuous TCR/tHLA interactions at 4°C might have been responsible
for these TCPF (9). Attempts to stain this PBMC population
at higher temperatures revealed that at room temperature is was still
possible to identify a tHLA-staining population. Staining carried at
37°C demonstrated almost a total loss of tHLA staining. However, it
could not be totally excluded that this loss of staining was due to
tHLA-induced down-regulation that can occur at this temperature, as
shown in Fig. 3. Indeed, marked reduction in intensity of tHLA staining
was noted in all PBMC populations when staining was carried at
37°C. Affinity of tHLA for vaccine-elicited T lymphocytes
Affinity of vaccine-elicited T cells for cognate epitope, rather
than their status of activation, might modulate their sensitivity to
stimulation. In fact, tHLA have been used to identify high avidity
tumor-reactive CTL (21). Therefore, intensity of tHLA
staining of vaccine-elicited T cells was compared with that of T cell
clones whose requirements for activation could be characterized (Fig. 5A). Epitope-driven CTL
cultures of postvaccination PBMC from earlier patients vaccinated with
g209-2 M were cloned according to previously described methods
(16, 17). The clones were then tested for ability to
produce IFN- in response to stimulation with T2 cells pulsed with
g209 (and 209-2 M, not shown) peptide. Among them, a clone was selected
for its poor avidity for the HLA-A*0201/g209 complex (C1–35), two for
intermediate avidity (C11 and C6), and two for increasingly high
avidity (B3 and H5). In addition, the TIL clone 1520 H-3.1 naturally
recognizing g209 and cross-reacting with g209-2 M was analyzed.
Staining for CD3 and tHLA was then compared among these clones and
tHLA+ PBMC from patients in this study (Fig. 5B). Most tHLA-positive PBMC stained for tHLA with intensity
comparable with that of intermediate affinity CTL and TIL (C6,
C11, and 1520 H-3.1). Although the expression of CD3 and CD8
coreceptors can play an important role in T cell activation/avidity
(22, 23), we did not notice large differences in
expression of these two markers in tHLA-staining PBMC in these
patients. Thus, differences and similarities of tHLA staining in these
patients could probably be attributed directly to avidity of the TCR
for the cognate ligand. Interestingly, in two patients (P4 and P7),
relatively lower intensity of staining was noted that correlated with a
reduced ability to detect IFN- upon stimulation by IC-FACS. In both
patients, however, qRT-PCR could demonstrate evidence of
vaccination-induced enhancement of IFN- expression upon cognate
stimulation. It is possible that this might reflect higher sensitivity
of this method compared with IC-FACS. Fluorescence of individual T
cells producing low amounts of IFN- might not be discriminated from
background fluorescence by FACS. At the same time, qRT-PCR might detect
low abundance transcript if a sufficient number of cells express,
although at low levels, IFN-. Vaccine-naive PBMC from P2 (P2-0) were
also included in this analysis. These PBMC demonstrated the lowest
intensity of staining that correlated with total unresponsiveness to
stimulation, strengthening the hypothesis that these PBMC promiscuously
bound g209-2 M/tHLA at the temperatures used in this study.
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PBMC from the same patients were assessed for ability to express
IFN- upon exposure to melanoma cells expressing the HLA allele
targeted by the vaccination (A*0201). In general, a good correlation
was noted between vaccine-epitope and tumor cell recognition whether
assessed by IC-FACS (Fig. 6) or by
qRT-PCR (Table II). However, surprisingly, tumor cells appeared to be
recognized more frequently after vaccination and independently of
documented vaccine-elicited T cell responses. This finding suggested
that vaccines (either the MA epitope or the IFA) might induce broader
immune effects than those strictly expected by the specificity of the
vaccination.
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In all patients (P3, P4, P5, P6, P7, and P8) in which enhancement
of TCPF was noted after vaccination, we tested the CD45 RO/RA phenotype
of vaccine-specific T cells. We found an unpredictable pattern of CD45
expression with a patient characterized by an almost totally CD45
ROhigh tHLA-staining population (P7) (Fig. 7A). A second patient (P8)
demonstrated a high frequency of CD45 RA/RO double-negative tHLA
staining T cells (Fig. 7B). Interestingly, in this patient,
the double-negative T cells appeared to be extremely sensitive to
cognate stimulation, as judged by the level of tHLA down-regulation
upon epitope stimulation. The other patients (P3, P4, P5, and P6)
demonstrated a pattern in between the other two patients with a small
proportion of tHLA-staining T cells included in all CD45 categories.
Thus, the correlation between reactivity of tumor-specific T cells and
their CD45 RO/RA phenotype noted by others (6, 7) was not
identified in this study, indicating that subsets of memory lymphocytes
with various effector function might be better resolved by other
markers (24).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Alternatively, vaccine-elicited responses identified by current methods might have not achieved in qualitative and/or quantitative terms the richness required for clinical effectiveness. Vaccine-elicited T cell responses have been predominantly characterized by indirect methods, including parallel in vitro sensitization of pre- and postvaccination PBMC with vaccine-related epitopes (1, 2). Although in vitro expansion of epitope-specific T cells indisputably demonstrated vaccine-related effects on vaccine recipients, it cannot enumerate and characterize the in vivo function of vaccine-elicited T cells because of the arbitrary amount of Ag and cytokine applied in culture.
Adopting tHLA technology, we directly documented and quantified the
immune response in PBMC of patients receiving the gp100-related
epitope: g209-2 M (8). We noted a correlation between
increased vaccine-specific TCPF and susceptibility to in vitro
stimulation. However, it was generally felt that such demonstration did
not adequately address the in vivo status of T cell activation
(7). In fact, the ability of tumor-specific circulating T
cells identified by tHLA to respond to physiologic levels of cognate
stimulation has been questioned, and selective anergy has been invoked
to explain T cell and tumor coexistence (7). Others have
attributed discrepancies between tHLA staining and T cell
responsiveness to promiscuity of TCR/tHLA interactions since the
enhanced affinity induced by multimerization of peptide/HLA complexes
may allow binding to TCRs with avidity too low to ever generate
ligand-induced physiological responses (9). We, therefore,
studied the level of activation of vaccine-elicited circulating
CD8+ T cells by combining a series of methods
that allow direct assessment of T cell function in PBMC with minimal ex
vivo manipulation and comparing these results with tHLA-based
phenotyping. These methods included measurement upon vaccine-specific
stimulation of IFN- transcript and protein by qRT-PCR or IC-FACS
(8, 10).
Originally, we hoped to identify tHLA/IFN- double-positive T cells
in vaccinated patients, but rapidly learned that such events cannot
occur. Staining of epitope-specific T cells was disproportionately
down-regulated when incubation with tHLA was performed at 37°C, but
not at 4°C. Lack of detection of
tHLA+/IFN--producing T cells was due to a
direct effect of TCR engagement in metabolically active cells
than tHLA internalization since it occurred upon cognate stimulation at
37°C, followed by tHLA staining at 4°C, a temperature at which
internalization of tHLA does not occur (9). tHLA
down-regulation could be observed in a g209 and g209-2 M-reacting T
cell clone characterized by good responsiveness to limiting
concentrations of epitope and naturally recognizing melanoma cells
(16). This down-regulation followed incubation with tHLA
at 4°C, following stimulation at 37°C with epitope-loaded T2 cells
or melanoma cells. Furthermore, down-regulation correlated with CD69
expression, suggesting that it is more likely related to the metabolic
and functional state of the cell rather than to artifactual tHLA/TCR
interactions at various temperatures. Thus, we concluded that
down-regulation of HLA staining is associated with T cell activation
and parallels TCR down-regulation (25). We, therefore,
considered down-regulation of tHLA staining additional evidence of
productive TCR/epitope engagements detectable in vaccine-specific
PBMC.
Because of tHLA-staining down-regulation, direct evidence of
vaccine-elicited T cell responsiveness to vaccine-related stimuli could
not be sought by identifying tHLA/IFN- double-positive T cells.
Thus, we evaluated responsiveness of PBMC by correlating tHLA
down-regulation with staining for IFN- protein and measurement of
IFN- transcript by qRT-PCR in pre- and postvaccination PBMC. IC-FACS
staining suggested responsiveness to stimulation with peptide in five
of the six patients who demonstrated vaccine-elicited increase in TCPF
by tHLA. qRT-PCR appeared more sensitive, as it could detect IFN-
transcript in all of them. Upon stimulation with tumor cells,
enhancement of IFN- production could be detected in all patients
independently of the method (IC-FACS or qRT-PCR), suggesting that
stimulation with tumor cells represented a stronger stimulus than the
exogenous administration of soluble peptide. Indeed, the direct
administration of soluble peptide to PBMC preparations may represent a
suboptimal stimulus that was purposefully chosen to approximate the
responsiveness of vaccine-elicited T cells to nonspecialized Ag
presentation. It was felt that stimulation of PBMC with T2 cell line or
other professional APCs that express abundant amounts of costimulatory
and adhesion molecules might have arbitrarily exaggerated the
responsiveness of vaccine-elicited T cells.
It has been shown that suboptimal activation of melanoma Ag-specific
lymphocytes could be related to low avidity of TCR/MHC tumor peptide
interaction (26). Such low avidity TCR may be included in
the repertoire of postvaccination PBMC. A limited g209-specific
TCR repertoire might be naturally available in vaccine-naive patients,
since gp100 is a self protein and elimination of high affinity TCRs has
recently been shown to play a role in these circumstances in another
tumor model (27). Furthermore, the vaccination itself
might have eliminated high avidity T cell clones
(28, 29, 30, 31), although such phenomenon is, at the moment,
controversial (32). In this study, tHLA-guided
identification of vaccine-specific T cells did not always correlate
with IFN- expression upon stimulation. Since it has been shown that
the avidity of TCR/MHC epitope interaction can be graded by measuring
the intensity of tHLA staining of tumor-specific T cells
(21), we evaluated this parameter in vaccine-elicited
PBMC. Assessment of tHLA staining of vaccine-elicited T cells in those
cases in which no IFN- induction was noted upon stimulation
suggested a lower avidity of their TCR compared with that of PBMC that
could clearly produce IFN- upon stimulation. Thus, lack of
correlation between tHLA-guided identification of vaccine-elicited T
cells and IFN- production appeared to be explainable by lower
avidity of the TCR repertoire induced by the vaccination in some
patients.
An additional finding of this study was the consistently larger number of T cells that could be identified with g209-2 M/tHLA compared with those that could be identified by IC-FACS upon stimulation. This finding emphasizes that tHLA staining may overestimate the frequency of functional Ag-specific CTL especially when, like in our case, tetramer-low cells are counted as tetramer positive (for instance, in patient P2-0) (9). Alternatively, a significant proportion of vaccine-specific T cells identified by tHLA could display blunted reactivity to antigenic stimulation. Whether the glass is half empty or half full remains an open question; however, the identification of a significant percentage of stimulation-responsive T cells in postvaccination PBMC suggests that anergy is not a global occurrence among vaccine-elicited T cells.
It has been noted that tHLA staining calculates CTL precursor frequencies considerably higher than those suggested by enzyme-linked immunospot or limiting dilution assays (33). Several studies have measured the response of T cells to acute or chronic viral infections or during ongoing autoimmune episodes (33, 34, 35, 36, 37, 38). Studies on tumor reactivity have shown that, in melanoma patients with vitiligo, MART-1-specific CTL have been identified at a frequency up to 0.67% of CD8+ T cells (39). Furthermore, MART-1-specific T cells could be identified in melanoma cell-infiltrated lymph nodes, with a frequency ranging from 0.22 to 1.8% of CD8+ T cells, and correlated with MA expression (40). Characterization of circulating T cells demonstrated identifiable MA-specific T cells in approximately one-half of patients affected with metastatic melanoma (7). In this study, vaccine-specific T cell frequency increased up to 2.3% of CD8+ cells. This is a relatively low number of CTL precursors after vaccination compared with viral and autoimmune models. An inverse correlation has been reported between HIV-specific CTL frequency and viral RNA load in HIV-infected individuals (34). Furthermore, TCPF as high as 2% of CD8+ T cells have been reported in HIV-infected patients, who remained asymptomatic (36). 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 rather that not being qualitatively adequate in terms of individual T cell responsiveness.
In this study, we could not easily attribute lack of tumor regression
after vaccination to anergy of circulating vaccine-elicited T cells.
Although possible that anergic tumor Ag-specific T cells may coexist
with growing tumor masses in naive patients, the same phenomenon does
not apply in vaccine settings. Considering our recent identification of
IFN--producing vaccine-elicited T cell responses at tumor site
(10) and the enhanced ability to induce vaccine-specific T
cells from identical lesions after vaccination (41), we
hypothesize that vaccine-elicited T cells coexist with tumor cells in
the tumor microenvironment. Thus, we blame the lack of clinical
effectiveness of MA-specific vaccines on still unknown quantitative
and/or qualitative limits of the T cell response within the tumor
microenvironment.
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Footnotes |
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2 Abbreviations used in this paper: MA, melanoma Ag; gp, glycoprotein; IC-FACS, intracellular FACS; qRT-PCR, quantitative real-time PCR; TCPF, T cell precursor frequency; tHLA, HLA/epitope tetrameric complex; TIL, tumor-infiltrating lymphocyte.
Received for publication March 7, 2000. Accepted for publication May 31, 2000.
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