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Kinetics of TCR Use in Response to Repeated Epitope-Specific Immunization

Vladia Monsurrò^*, Mai-Britt Nielsen, Ainhoa Perez-Diez, Mark E. Dudley, Ena Wang, Steven A. Rosenberg and Francesco M. Marincola^1,*,

^* HLA Laboratory, Department of Transfusion Medicine, Clinical Center, and Surgery Branch, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Selection of T cell-directed immunization strategies is based extensively on discordant information derived from preclinical models. We characterized the kinetics of T cell selection in response to repeated antigenic challenge. By enumerating with epitope/HLA tetrameric complexes (tHLA) vaccine-elicited T cell precursor frequencies (Tc-pf) in melanoma patients exposed to the modified gp100 epitope gp100:209–217 (g209-2M) we observed in most patients that the Tc-pf increased with number of immunizations. One patient’s kinetics were further characterized. Dissociation kinetics of g209-2M/tHLA suggested enrichment of T cell effector populations expressing TCR with progressively higher affinity. Furthermore, vaccine-elicited T cells maintained the ability to express IFN- ex vivo and proliferate in vitro. Thus, repeated exposure to immunogenic peptides benefited immune competence. These results provide a rationale for immunization strategies.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The identification of melanoma-associated Ags (MAA)² and their T cell epitopes has stimulated interest in the development of active specific vaccination of patients with advanced cancer (1, 2). The strategies adopted for these immunizations have varied significantly, and their selection has been based mainly on assumptions derived from preclinical models or on empirical estimates of the most efficient compromise between practical and immunological choices (3, 4, 5). Comparison of clinical and immunologic results from these pivotal studies has shown a complex array of often-contradictory information. Short-term epitope-specific vaccination against gp100/PMel17 administered in IFA was highly effective in eliciting systemic T cell responses but had limited clinical effectiveness in the absence of concomitant IL-2 administration (3). Repeated long-term administration of melanoma Ag gene-derived peptides induced clinical responses associated with minimal evidence of systemic T cell activation (4). Administration of APCs loaded with peptide epitopes could induce clinical response and immune activation, although the immunologic parameters presented were limited and difficult to generalize (5). Thus, in the absence of a correlation between clinical and immunologic end points, the rationale for the selection of immunization schedules is not defined. Furthermore, the kinetics of the immune response to repeated exposure to HLA class I-associated epitopes is unknown.

Preclinical studies suggest that repeated exposure to limited epitope sequences may tolerize or sensitize animals against a given Ag depending on the identity of the peptide used, the route of administration, or the carrier used (6, 7). Others have suggested that the timing, frequency, and length of immunization might play a key role in achieving a threshold necessary for tumor regression (8, 9). Savage et al. (10) has shown that repeated immunization of mice with pigeon cytochrome c protein shifted the T cell repertoire selection toward T cells with higher affinity TCR/epitope interactions, suggesting that repeated vaccination might progressively benefit host immune competence. Based on these observations and recognizing that little is known about the kinetics of T cell repertoire selection in humans, we followed the immune response to sequential boosting with MAA-derived T cell epitopes by enumerating changes in T cell precursor frequency (Tc-pf) with HLA/epitope tetrameric complexes (tHLA; Ref. 11). Patients with metastatic melanoma who received vaccination with the gp100 epitope gp100:209–217(210M) (abbreviated here as g209-2M) in IFA were separated in two cohorts according to the frequency of this administration (weekly vs every third week). Tc-pf in PBMC were enumerated at different intervals. In a patient in whom sufficient reagents were available, functional and genetic characterization of the vaccine-elicited immune response was performed by linking Tc-pf to variation in epitope/TCR ligand affinity, responsiveness to cognate stimulus, and TCR V use.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Patient selection

Nine HLA-A*0201-expressing patients with metastatic melanoma received repeated s.c. injections of the g209-2M peptide in IFA. These were the only patients available for the analysis. Protocols had been approved by the Institutional Review Board of the National Cancer Institute and informed consent was obtained before the patient’s enrolment. Vaccinations were administered either at 1- or 3-wk intervals, and blood samples for PBMC extraction were obtained just before the next vaccination and 3 wk after the prior vaccination. None of the patients received any concomitant treatment including systemic IL-2. The HLA class I phenotype of patients was determined on PBMC by using sequence-specific primer-PCR (12). Because of the limited amount of clinical data-points available, the power of the analysis was not deemed sufficient for a statistical analysis. Thus, these results should be seen as exploratory only.

Cells and culture conditions

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 (CM) 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.

PBMC also were analyzed after 10 to 11 days of in vitro culture after stimulation with exogenous peptide (g209 or g209-2M). This was achieved by the addition of 1 µM peptide in CM 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.

Development of T cell clones

Bulk CTL cultures were cloned by limiting dilution according to a modification of Riddell’s technique (13, 14). 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). Clones used in this study (P1G9 and P1C3 from the patient belong to the weekly schedule of vaccination that reach the 16i) were selected after an in vitro expansion of the 12i time point according to their reactivity against HLA-matched, MAA-expressing tumor targets. Clones were further expanded as described previously (13).

Peptides

The peptides used for vaccination were prepared according to Good Manufacturing Practice by Multiple Peptide Systems (San Diego, CA). Peptides used for tHLA synthesis and in vitro stimulation studies were commercially synthesized by Princeton Biomolecules (Columbus, OH). The peptides were purified by gel filtration, and their identity was confirmed by mass spectral analysis. Peptide sequences are described below as relevant.

Epitope-specific T cell staining with tHLA

tHLA complexes were produced as described previously (11, 15). Recombinant HLA-A*0201 heavy chain containing a biotinylation site and recombinant ₂-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 parental gp100:209–217 (ITDQVTCPFSV, g209) and for the modified epitope for the vaccine gp100:209–217(210M) (IMDQVTCPFSV, g209-2M); monomeric HLA/peptide complexes were biotinylated with BirA (Avidity, Denver, CO) and tetramerized by adding avidin-PE (Pierce, Rockford, IL). After overnight depletion of monocytes or after 10–11 days of in vitro sensitization (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 µg of tHLA for 15 min, then continued for 30 min with 10 µl anti-CD8 mAb (100 µg/ml, Becton Dickinson, San Jose, CA). Cells were washed in 2 ml of FACS buffer and analyzed by FACS (Becton Dickinson). Ten 1,000 events were acquired for analysis of T cell clones or CTL after IVS and 200,000 for PBMC samples. 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 = upper right quadrant (URQ)/(URQ+lower right quadrant (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 (f_c). The f_c is presented as the number of peptide-specific T cells per 10⁶ CD8⁺ T cells.

For sorting, fresh PBMC and CTL after IVS were incubated at room temperature with 1 µg tHLA/10⁶ cells for 1 h then continued for 30 min with 10 µl anti-CD8 mAb/10⁶ cells (100 µg/ml, Becton Dickinson). Cells were washed in 2 ml of FACS buffer and sorted by SORT-Star-plus (Becton Dickinson).

FACS analysis for intracellular density of IFN-

Nonadherent PBMC (10⁶ cells) were stimulated for 6 h by directly adding soluble peptide (1 µg/ml). After 2 h, brefeldin A (BFA; 10 µg/ml; Sigma, Deisenhofen, Germany) was added. After an additional 4 h, cells were stained with PE-tHLA and PerCP-conjugated mouse anti-human-CD8 as described previously and fixed with 4% paraformaldehyde for 5 min. After wash in 0.1% BSA/PBS, cells were permeabilized and blocked overnight with PBS/saponin/5% milk at 4°C. After staining with FITC-conjugated mouse anti-human-IFN- for 30 min at 4°C, samples were analyzed on a Becton Dickinson FACSCalibur flow cytometer with the CellQuest software. Gating on CD8⁺ lymphocytes was performed during analysis.

Dissociation kinetics of g209-2M/tHLA binding to Ag-specific T lymphocytes

The cells were stained at 4°C with PE-conjugated tHLA (1 µg/10⁶ cells) for 45 min and 30 min with FITC-conjugated anti-CD8. Cells were washed two times and resuspended in 100 µl of FACS buffer. After the first time point, saturating concentrations of the HLA-A*0201-specific Ab BB7.2 was added (100 µg/ml). Fluorescence was assessed at 0 and 1 h. For all analyses, >50,000 events were collected and analyzed by the CellQuest program and expressed as normalized total fluorescence (the sum of the fluorescence intensity within the tetramer gate normalized per CD8 cells) as suggested previously (10).

RNA isolation, cDNA synthesis, and RNA amplification

RNA isolation, cDNA synthesis, and RNA amplification procedures were performed in batches containing all patient samples to minimize variability. After thawing the specimens, total RNA was isolated with RNeasy Mini kits (Qiagen, Chatsworth, CA) and quantified by spectrophotometer (GeneQuant, Clamart Cedex, France). Antisense RNA (aRNA) amplification was prepared according to previous description (16) with modification. Briefly, 1 µg of total RNA isolated from each sample was heated to 70°C for 3 min in the presence of 0.25 µg of oligo-dT T7 primer (5'-AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC-3'). Reverse transcription was conducted in 20 µl of reaction mixture with 0.5 µg of template switch primer (5'-AAG CAG TGG TAT CAA CGC AGA GTA CGC GGG-3'; Clontech, Palo Alto, CA), 4 µl first-strand buffer, 2 µl of 0.1 M DTT, 1 µl of RNAsin, 2 µl of 10 mM dNTP, and 2 µl of Superscript II at 42°C for 1 h. Full-length double-strand (ds) cDNA syntheses was initiated by template switch primer in the presence of 15 µl of Advantage PCR buffer (Clontech), 3 µl of 10 mM dNTP, 1 µl of RNase-H (Promega, Madison, WI), and 3 µl Advantage cDNA polymerase (Clontech) in a 150-µl volume and cycled at 37°C for 2 min, 94°C for 3 min, 65°C for 3 min, and 75°C for 30 min. The reaction was terminated by incubation with 7.5 µl of 1 M NaOH with 2 mM EDTA solution at 65°C for 10 min. ds cDNA then was purified by phenol-chloroform-isoamyl extraction and ethanol precipitation in the presence of 0.1 µg of linear acrylamide (Ambion, Austin, TX) and further by size permeation chromatography (Bio-6 column; Bio-Rad, Cambridge, MA). After lyophilizing ds cDNA was resuspended in 8 µl of diethyl pyrocarbonate-treated H₂O and in vitro transcribed into aRNA at 37°C for 5 h in a 20-µl reaction volume with a T7 Megascript kit (Ambion) according to manufacture manual. aRNA recovery was achieved by using Trizol reagent (Life Technologies). For the second round amplification 1 µg of aRNA from each sample mixed with 4 µg of random hexamer (Life Technologies, Rockville, MD), 1 µg of oligo dT-T7 primer, 4 µl of first-strand buffer, 2 µl of 0.1 M DTT, 1 µl of RNasin, 2 µl of 10 mM dNTP in a 20-µl volume was heated to 65°C for 10 min and then reverse-transcribed into anti-sense cDNA with 2 µl of Superscript II at 42°C for 1 h. ds cDNA synthesis and the following purification and in vitro transcription was performed as the first round. Reverse-transcription reaction from both tRNA and aRNA was accomplished by adding 2 µg of random hexamers, 4 µl of first-strand reaction buffer, 2 µl of 0.1 M DTT, 2 µl of 10 mM dNTP, and 2 µl of Superscript II at 42°C for 1 h. cDNA was stored at -20°C until use.

Quantitative real-time PCR

Levels of IFN- gene expression was assessed by real-time quantification assay based on TaqMan ABI Prism 7700 Sequence Detection System (Perkin-Elmer, Foster City, CA; Ref. 17). The number of target gene copies was extrapolated from a standard curve equation generated with known amounts of pure cDNA for each gene and normalized over the number of CD8 transcript. Probe and primers were designed to span intron-exon junctions to prevent amplification of genomic DNA and to enhance PCR efficiency by obtaining amplicons less then 150 bp in length. TaqMan probe were labeled at the 5' end with the reporter dye (molecule) 6-FAM (6-carboxyfluorescein; emission max = 518 nm) and at the 3' end with the quencher dye (molecule) TAMRA (6-carboxytetramethylrhodamine; emission max = 582 nm). Quantitative PCR of both specimen cDNA and standard amplified cDNA were conducted in a 25-µl final volume mixture containing primers, probe, and 1x TaqMan Master Mix (Perkin-Elmer) at optimized concentrations (probe, 200 nM; primers, 400 nM). 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 and annealing/extension at 60°C. Probe and primers sequences have been described previously (17).

TCR V PCR and clone-specific PCR

A set of 35 primers was selected to amplify 45 functional V (18). Each primer mix was composed of 10x PCR buffer, 1.5 mM MgCl₂, 200 µM dNTP, 1.25 U of AmpliTaq Gold, 0.5 µl of cDNA, 0.5 µM V primer, 0.5 µM TC-1 constant region primer, and water up to 20-µl final reaction volume. PCR was run with the following protocol: initial activation of the enzyme at 94°C for 9 min; 10 high-stringency cycles of 94°C for 30 s of denaturation, 65°C for 1 min of annealing, and 72°C for 1 min of elongation; 35 low-stringency cycles of 94°C for 30 s, 60°C for 1 min, and 72°C for 1 min; final extension at 72°C for 10 min. After PCR, 6 µl of the product and 3 µl of loading buffer were mixed and run on a 1% agarose gel for 45 min at 150 V. The gel was stained with Vistra Green (Amersham Life Science, Arlington Heights, IL).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Kinetics of T cell expansion in response to repeated epitope-specific immunization

Repeated exposure to HLA class I epitope was studied in two similar patient protocols. In the first, four HLA-A*0201-expressing patients with metastatic melanoma received weekly s.c. injections of 1 mg of 209-2M peptide emulsified in IFA. This peptide represents an anchor residue modification from the wild-type gp100/PMel17 glycoprotein epitope gp100:209–217(g209) (19). In the second protocol, five HLA-A*0201-expressing patients with metastatic melanoma received s.c. injections of 1 mg of g209-2M peptide in IFA at 3-wk intervals. Tc-pf changes in response to vaccination were estimated by enumerating CD8 and tHLA/g209-2M (vaccine-specific; Fig. 1) or tHLA/g209 (tumor-specific; Fig. 2) double-positive PBMC. Interestingly, two populations of tetramer-staining cells were observed, one characterized by dim and the other by bright CD8 staining. Although we have noted recently that the dim CD8-staining population expresses a higher level of IFN- on cognate stimulation, it is not clear at the moment the significance of this phenomenon. Repeated vaccination induced dramatic enhancement in Tc-pf of vaccine-specific CD8⁺ T cells (Fig. 3). The enhancement in vaccine-elicited T cells was associated with an enhancement of Tc-pf specific for the wild-type epitope expressed by the gp100 glycoprotein and naturally recognized by vaccine naive T cells as shown in previous studies (20). Comparison of patients treated with the two schedules of administration suggested a faster expansion of g209-2M-specific Tc-pf with the weekly schedule. In addition, increases in Tc-pf seemed to correlate with number of vaccinations at least in patients that received the weekly vaccination. However, the increase in parental epitope-specific Tc-pf in relation to the number of vaccinations was less pronounced.

View larger version (43K): [in this window] [in a new window]   FIGURE 1. Kinetics of expansion of vaccine-specific CD8+ T cells in response to repeated vaccination. Enumeration of CD8 and tHLA/209-2M double-positive PBMC in response to repeated s.c. injections of g209-2M peptide in IFA. PBMC from aphereses obtained before any treatment and after 4, 8, 12, and 16 vaccinations were frozen after Ficoll separation. The samples then were thawed and concomitantly tested at the end of the treatment course. Staining was performed with FITC-conjugated anti-CD8 mAb and PE-labeled tHLA/g209-2M. In each experiment, 200,000 events were analyzed per sample. The figure is representative of three experiments with similar results performed in the same patient as in Fig. 2. All patients’ PBMC were similarly analyzed by direct ex vivo phenotyping as well as after one round of in vitro stimulation. Tc-pf were analyzed directly in thawed PBMC and T cell cultures elicited from PBMC with one in vitro stimulation with g209 or g209-2M peptide (1 µM) and 300 IU IL-2. Percentage of tetramer-staining CD8+ T cells is shown in the left upper quadrant of each experiment.

View larger version (43K): [in this window] [in a new window]   FIGURE 2. Kinetics of expansion of gp100 wild-type epitope-specific CD8+ T cells in response to repeated vaccination. Enumeration of CD8 and tHLA/209 double-positive PBMC in response to repeated s.c. injections of g209-2M peptide in IFA. PBMC from aphereses obtained before any treatment and after 4, 8, 12, and 16 vaccinations were frozen after Ficoll separation. The samples then were thawed and concomitantly tested at the end of the treatment course. Staining was performed with FITC-conjugated anti-CD8 mAb and PE-labeled tHLA/g209. In each experiment 200,000 events were analyzed per sample. The figure is representative of two experiments with similar results performed in the same patient as in Fig. 1. All patients’ PBMC were similarly analyzed by direct ex vivo phenotyping as well as after one round of in vitro stimulation. Tc-pf were analyzed directly in thawed PBMC and T cell cultures elicited from PBMC with one in vitro stimulation with g209 or g209-2M peptide (1 µM) and 300 IU IL-2. Percentage of tetramer-staining CD8+ T cells is shown in the left upper quadrant of each experiment.

View larger version (26K): [in this window] [in a new window]   FIGURE 3. Expansion of vaccine-specific and wild-type-specific T cells in response to repeated exposure to g209-2M. Vaccine-specific T cells were enumerated by using g209-2M/tHLA (g209-2M Tc-pf) and natural epitope-specific T cells by using g209/tHLA (g209 Tc-pf). Tc-pf values are expressed as number of tHLA-staining T cells/106 CD8+ T cells. Tc-pf were analyzed directly in PBMC (PBMC) and in 10-day CTL cultures induced by in vitro stimulation with the vaccine (g209-2M) or the parental (g209) epitopes. PBMC and cultures from four patients who received weekly vaccination are shown as solid symbols and those from five patients treated at 3-wk intervals are shown as empty symbols.

To functionally characterize vaccine-elicited T cells, PBMC obtained before and after 4, 8, and, when available, 12, and 16 vaccinations also were expanded in vitro by a single exposure to g209-2M or g209 peptide (1 µM) and 300 IU/ml IL-2 (Figs. 1 and 2). In this way, the responsiveness of vaccine-elicited T cells to in vitro stimulation could be correlated with tHLA-derived Tc-pf. Ten days after stimulation, frequencies of tHLA-staining CD8⁺ T cells were enumerated. The rapid expansion of vaccine-specific T cells observed in patients who received weekly vaccination corresponded to a significant difference in Tc-pf calculated after in vitro expansion of PBMC whether the expansion was induced by stimulation with the vaccine-related peptide g209-2M or the parental peptide g209 (Fig. 3). In most cases, vaccine-specific T cell increased rapidly on stimulation in vitro with either modified or parental epitope, and their expansion was highly correlated with the Tc-pf in the originating PBMC population. Similarly, g209-specific T cells retained responsiveness to either peptide and their kinetics of expansion in vitro were similar to those of g209-2M-specific T cells independent of the epitope used for the secondary in vitro stimulation. Thus, this preliminary analysis of the kinetic response to repeated peptide exposure excluded depletion of epitope-specific T cells and, on the contrary, suggested a relative expansion with time.

Modulation of TCR affinity in response to repeated Ag encounters

To characterize the kinetics of expansion of vaccine-induced T cells, we followed a patient who had completed 16 weekly vaccinations (, Fig. 3). The effect of repeated vaccination on vaccine-specific immune competence was tested functionally by enumerating with intracellular (IC)-FACS vaccine-specific T cells that could respond to stimulation with g209-2M (Table I). CD8⁺ T cells were gated and analyzed for staining with tHLA/g209-2M (Fig. 4A) after stimulation with 0.1 µM peptide. As noted previously (21), epitope stimulation induced down-regulation of TCR staining with tHLA that correlated with identification of IFN--producing T cells. This could be clearly seen in a pure population of epitope-specific effectors represented by P1C3 CTL clone expanded from PBMC of the same patient after 12 immunizations. The number of IFN--producing T cells increased with the number of vaccinations (Fig. 4B), and this increase appeared to parallel the overall increase in Tc-pf. The ratio of IFN--producing cells over the number of tHLA/g209-2M staining T cells did not substantially change with time and remained around 10% of the tetramer staining cells. The overall responsiveness to the vaccine epitope also was assessed by quantitative real-time PCR by stimulating PBMC with limiting dilutions of g209-2M and g209 and testing for IFN- transcript level normalized by CD8 (17). Also, in this case, the cumulative responsiveness of PBMC toward the peptide used for vaccination increased with number of vaccinations (Fig. 4C). Although less intense, the reactivity toward the wild-type g209 epitope also increased. Interestingly, we noted that the concentration of g209-2M and g209 peptide necessary to elicit epitope-specific expression of IFN- in PBMC decreased to 1 and 10 nM, respectively, after the 12th vaccination.

View this table: [in this window] [in a new window]   Table I. IFN- transcript abundance in response to ex vivo stimulation with vaccine-related epitopes

View larger version (22K): [in this window] [in a new window]   FIGURE 4. Functional characterization of the kinetics of vaccine-elicited T cell responses. A, A g209-2M-specific CD8+ T cells clone (P1C3) expanded after 12 immunizations and PBMC obtained after 4 (4i) and 16 immunizations (16i) were stimulated in vitro at 37°C with g209-2M peptide (0.1 µM) and the frequency of IFN--producing CD8+ T cells enumerated by IC-FACS. Stimulated (g209-2M) and unstimulated (no stim) T cell populations were gated according to staining with CD8-PerCP mAb and evaluated for tHLA/g209-2M (PE) surface and IFN- (FITC) intracellular staining. On cognate stimulation, tHLA staining is down-regulated as discussed in a previous paper (21 ). B, Results of a complete set of experiments in which PBMC obtained after 4, 8, 12, and 16 immunizations (4i to 16i) were tested for IFN- production by IC-FACS as demonstrated in A. Data are shown for unstimulated PBMC () and PBMC stimulated with 0.1 µM g209-2M () as number of T cells/106 CD8+ T cells. C, Quantitative real-time PCR-based assessment of IFN- transcript production in response to stimulation of PBMC with increasing concentrations of g209-2M peptide (from 0 to 10 µM). PBMC were obtained after 4, 8, 12, and 16 immunizations as in B, thawed concomitantly, and stimulated with peptide after overnight resting in CM without additional cytokines (17 ). Transcript abundance is expressed as number of IFN- mRNA copies/104 of CD8 mRNA copies.

View larger version (11K): [in this window] [in a new window]   FIGURE 5. Kinetics of tHLA staining decay. PBMC obtained after 4, 8, 12, and 16 immunizations were thawed concomitantly and rested overnight in CM without additional cytokines. PBMC then were stained at 4°C with tHLA/g209-2M (PE) and CD8 mAb (FITC) in the presence of the HLA-A*0201-specific BB7.2 mAb to capture dissociated tHLA complexes. Fluorescence was assessed at time 0 and after 1 h. The total PE fluorescence within the tetramer gate (mean PE fluorescence time number of events in the tetramer gate) then was normalized for each data point per total CD8 cells (whether tHLA-positive or -negative) as discussed previously (10 ). The fluorescence decay plots illustrate the percent of total PE fluorescence at various experimental time points with time 0 arbitrarily considered 100%. A, Data obtained in PBMC. B, Data obtained in CTL cultures exposed to one in vitro stimulation with 1 µM g209-2M followed by 10 days of culture in CM with 300 IU/ml IL-2. C, Data obtained by using two clones expanded from limiting dilution of PBMC obtained after 12 immunizations. TCR V-chain usage was V6s2 (Fig. 6, lane 34) and V17s1 (Fig. 6, lane 14) for clone P1C3 and P1G9, respectively. The figure is representative of two experiments with similar results.

We then evaluated the genetics of TCR use in response to vaccination by sorting double-positive tHLA/g209-2M and CD8 T cells in PBMC, from the same patients, obtained after 4, 8, 12, and 16 vaccinations (Fig. 6A). With this strategy, it was possible to obtain >90% pure populations of tHLA-staining T cells according to a high-stringency gate. Total RNA then was prepared from the sorted PBMC and amplified into anti-sense RNA according to a previously validated procedure (16) to obtain sufficient material for V utilization analysis. Antisense RNA then was converted into cDNA for analysis of V-chain use. Only a few V-chains could be identified in the sorted CD8/tetramer double-positive PBMC populations. Most V-chains were transient, as they only were identified in one or two time points, suggesting that TCR repertoire expansion in response to repeated epitope-specific immunization is not attributable to an expanding clonal population. However, exceptions could be noted, as for V-8s1, 2 (lane 6, Fig. 6B) that appeared after eight immunization and persisted for the duration of the study. Because of the limited material obtainable from tHLA-sorted PBMC, we also expanded PBMC populations in vitro by exposing them to 1 µM g209-2M followed by 10 days of culture in the presence of IL-2 (300 IU/ml). In this way, a larger population of vaccine-specific T cells was obtained. These CTL cultures then were sorted according to tetramer staining (> 95% CD8/tHLA double-positive T cells) and tested for V-chain use without further amplification. This procedure increased the sensitivity of the analysis, as more V-chains could be amplified. Interestingly, the overall number of vaccine-specific V-chains identified with this method increased with the number of vaccinations, suggesting that repeated epitope-specific immunizations increase, rather than limit, the TCR repertoire. Interestingly, only one of the two CTL clones derived from 12i PBMC expressed the V-chain identified in the PBMC population (P1G9 expressing V 17s1; Fig. 6B, lane 14) whereas in the other case it did not (P1C3 expressing V 6s3, 4 or 6; Fig. 6B, lane 33). Both V-chains were identifiable in tetramer-positive T cells after epitope-driven in vitro expansion. Because both clones demonstrated lower dissociation rates than the average rates of tetramer-positive 12i PBMC, it is possible that their selective identification marked the preferential expansion in vitro of T cell-bearing high-affinity TCRs.

View larger version (55K): [in this window] [in a new window]   FIGURE 6. Kinetics of TCR use in vaccine-specific T cells. A, 108 PBMC obtained after 4, 8, 12, and 16 immunizations (4i to 16i) were thawed and rested overnight rest in CM in the absence of any cytokine. Nonadherent cells then were double-stained with tHLA/g209-2M (PE) and anti-CD8 mAb (FITC). Double-positive cells were sorted according to the gates shown in the scatter plots including tetramer-positive/CD8-positive (right upper quadrant) and tetramer-negative/CD8-positive (right lower quadrant) T cells. To avoid overlap of T cell populations, a gap was arbitrarily set between the two gates. In the second column of the scatter plot, the yield and purity of double-positive T cells is shown for each PBMC population (no. of cells = total number of viable double-positive cells obtained from each PBMC population). The percentages in the figure illustrate the number of T cells that after sorting were included in the "tetramer-positive gate." B, Total RNA then was prepared from sorted cells, and the material obtained was amplified with two rounds of amplification into aRNA as described previously (16 ). The fidelity of this method for unbiased amplification of various V-chains was tested by using pooled PBMC populations (data not shown). aRNA from sorted PBMC then was converted into cDNA as template for V utilization analysis with previously described primers (18 ). The same procedure used for PBMC was used after IVS of PBMC with 1 µM g209-2M followed by 10 days of expansion in CM with 300 IU/ml of IL-2. V use is shown for sorted PBMC populations (4i through 16i), for CTL lines expanded in vitro from the respective PBMC (4iIVS through 16iIVS) and for two CTL clones (P1G9 and P1C3) expanded from 12iIVS. At the top of the gel is the lane number. V-chain identity of amplicons from various lanes corresponds as previously described (18 ) to: lane 1, V 12s1, 12s2; lane 2, V 12s3; lane 3 V 21s1; lane 4, V 21s3; lane 5, V 21s2; lane 6, V 8s1, 8s2; lane 7, V 8s3; lane 8, V 23s1; lane 9, V 13s1, 13s2; lane 10, V 16s1; lane 11, V 24s1; lane 12, V 25s1; lane 13, V 18s1; lane 14, V 17s1; lane 15, V 22s1; lane 16, V 15s1; lane 17, V 11s1; lane 18, V 14s1; lane 19, V 3s1; lane 20, V 4s1; lane 21, V 2s1; lane 22, V 9s1; lane 23, V 20s1; lane 24, V 7s1; lane 25, V 7s2, 7s3; lane 26, V 5s1; lane 27, V 5s2, 5s3, 5s6; lane 28, V 5s4; lane 29, V 13s3; lane 30, V 13s5; lane 31, V 13s6; lane 32, V 6s1, 6s5; lane 33, V 6s3, 6s4, 6s6; lane 34, V 6s2; and lane 35, V 1s1.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Savage et al. (10) suggested in a murine model that repeated immunization selectively expands T cells expressing TCR with higher affinity for the immunogen applied. The present study is in agreement with this preclinical model, demonstrating that repeated exposure to the same epitope enhances the host immune competence by increasing the frequency of immunogen-specific precursor T cells, by selecting higher affinity TCR, and possibly by broadening TCR repertoire use. Although closer (weekly) immunizations appeared to induce faster T cells responses, the number of exposures also had a positive influence on Tc-pf. This finding, if corroborated by a larger patient series, might have strong practical implications in the field of tumor immunology, providing a rationale for the timing of immunization strategies. In fact, the limited clinical effectiveness of Ag-specific vaccines might be explained by an insufficient expansion below a threshold necessary for tumor rejection of effector T cell populations with present administration schedules. Response to acute or chronic viral infection, or in the context of autoimmune episodes, can drive an overwhelming immune response toward the pathogen or self-Ag (10, 29, 30, 31, 32, 33, 34, 35, 36). For instance, Butz and Bevin (29) have shown that at least 25% of CD8⁺ T cells produce IFN- in response to stimulation with lymphocytic choriomeningitis virus after a single in vivo exposure to this pathogen. In humans, Tc-pf of up to 44% of circulating CD8⁺ T cells have been noted during acute EBV infection (34). However, minimal information is available about the effect of vaccination of human subjects with minimal CTL epitopes, and most of this experience is restricted to anticancer studies. Most immunizations against cancer that have used minimal epitopic sequences have limited the number of vaccinations to few exposures for practical reasons, and they have so far demonstrated limited induction of vaccine-specific T cells. In a previous study (11), we noted that anticancer vaccine-specific Tc-pf reached only a maximum of 2.5% of circulating CD8⁺ cells. In this study, we used available human reagents from patients that had received repeated immunizations in the absence of other concomitant treatment.

We showed that even after 12 immunizations, vaccine and wild-type-specific Tc-pf reach a maximum of 48,000 and 37,000 per million CD8⁺ T cells, respectively (5 and 4%; Fig. 3) and only 10% of the tHLA-staining T cells express IFN- after exposure to the vaccine epitope ex vivo. Thus, the number of tumor-reactive T cells induced by the vaccine might be orders of magnitude below the number of effector T cells reportedly effective in clearing viral infections.

It has been suggested that in vaccine-naive patients, blunting of the status of activation of cancer-reactive T cells might be responsible for their coexistence with tumor cells in the cancer-bearing host (37). However, we recently have analyzed extensively the function of vaccine-elicited T cells by assessing the capacity of circulating T cells to respond to vaccine stimulation as well as HLA-matched tumor targets (21). In this study, we further tested this hypothesis by combining functional studies with studies evaluating TCR use. Functional studies indicated that the number of T cells producing IFN- in response to epitope-specific stimulation ex vivo is generally lower than that predicted by tHLA staining of the same PBMC population. Furthermore, V use studies suggested that although dominant V-chains could be identified in tHLA-sorted PBMC, these did not invariably correspond to V detectable after IVS. This suggests that circulating T cell populations capable of binding with tHLA are not necessarily responsive to ex vivo stimulation and in a significant proportion are incapable of proliferation after exposure to cognate epitope.

Repeated antigenic challenge results in enhanced immune response through the establishment of immunological memory. However, the relationship between the development of immunological memory and TCR selection remains unclear. Because T cells do not undergo somatic hypermutation to achieve high ligand affinity, the identity of a particular T cell population can be recalled through characterization of the clonality of their TCR. However, the findings of Savage et al. (10) suggest that selection of high-affinity TCR enhances immunological memory during the course of a protracted antigenic challenge (38). Similarly, in this study, we noted that in humans, repeated stimulation restricted to a single peptide/HLA class I allele combination induces an enhancement of the immune competence associated with an overall increased affinity of TCR ligand interactions. This selection of T cells could not be easily tracked to the oligoclonal expansion of T cell clones bearing a specific TCR V as noted in a murine infectious disease model (39). However, our inability to identify an oligoclonal expansion of specific T cells does not negate the kinetic signaling (40) and TCR clustering model (41) of T cell activation as the basis of T cell selection by repeated Ag exposure. In fact, this study is in line with the finding of Savage et al. (10) suggesting that successive antigenic exposure selects and expands Ag-specific T cells expressing TCRs with lower dissociation rates for peptide-MHC ligand. However, in this human model, a new level of genetic complexity is suggested in which new T cell populations, rather than oligoclonal expansion (42, 43) emerge during time to sustain the immune response.

It seems that only a small proportion of tHLA binding T cells maintain at a single time point the ability to react with the cognate epitope. Furthermore, expansion of epitope-specific T cells in vitro does not necessarily yield the T cell populations predicted by V-chain analysis of PBMC. However, the finding that there was not continuity noted in V use (Fig. 6B), may introduce a conceptual bridge between the TCR clustering model of clonal expansion (10) and clonal exhaustion (25). Refurbishing of novel effector T cells characterized by a higher average affinity for the immunogenic stimulus appears to operate in association with the disappearance of other T cells population as suggested by the fading of T cell clones with specific V-chains throughout time (Fig. 6B, lanes 15, 18, and 24). It is not easy to explain how new high-affinity circulating T cells are triggered only at such distant time from the primary exposure. It is conceivable that frequency of high-avidity T cells is much lower than that of low-avidity T cells in the context of self-Ags such as gp100 (44). This low frequency may require a large number of antigenic administrations before productive TCR/ ligand interactions may occur by chance. Our findings also are consistent with the lymphocytic choriomeningitis virus infectious model in mice where on persistent infection with high viral load anergy occurs with some and not other epitopes and, ultimately, deletion of specific T cell populations is observed (45). It is possible that the tumor load, besides the repeated vaccination, could narrow the vaccine-induced TCR repertoire. In this case, continuous modulation of the TCR repertoire could be expected as noted in this study.

In summary, this study suggests that, as predicted by murine models, protracted exposure to Ag stimulation has beneficial effects on the selection and expansion of T cell competence. The outmost potential of repeated immunogenic exposures may not be reached even after 12 immunizations, and future studies in humans should address this potential with particular attention to its clinical implications.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Francesco M. Marincola, Surgery Branch, National Cancer Institute, Building 10, Room 2B42, 10 Center Drive, MSC 1502, Bethesda, MD 20892-1502.

² Abbreviations used in this paper: MAA, melanoma-associated Ags; Tc-pf, T cell precursor frequency; tHLA, HLA/epitope tetrameric complexes; g209-2M, gp100 epitope gp100:209–217(210M); CM, complete medium; aRNA, antisense RNA; ds, double strand; IVS, in vitro sensitization; IC, intracellular; URQ; upper right quadrant; LRQ, lower right quadrant.

Received for publication December 18, 2000. Accepted for publication February 21, 2001.

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