Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0104026 on July 7, 2004

Published online before print July 7, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0104026v1
76/4/787    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Leggatt, G. R.
Right arrow Articles by Frazer, I. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Leggatt, G. R.
Right arrow Articles by Frazer, I. H.
(Journal of Leukocyte Biology. 2004;76:787-795.)
© 2004 by Society for Leukocyte Biology

Changes to peptide structure, not concentration, contribute to expansion of the lowest avidity cytotoxic T lymphocytes

Graham R. Leggatt1, Sharmal Narayan, Germain J. P. Fernando and Ian H. Frazer

Centre for Immunology and Cancer Research, University of Queensland, Princess Alexandra Hospital, Brisbane, Australia

1Correspondence: CICR, 4th Floor, Research Extension, Building 1, Princess Alexandra Hospital, Woolloongabba, Queensland, Australia, 4102. E-mail: gleggatt{at}cicr.uq.edu.au


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The efficient in vitro expansion of antigen-specific CD8+ cytotoxic T lymphocytes (CTL) for use in adoptive immunotherapy represents an important clinical goal. Furthermore, the avidity of expanded CTL populations often correlates closely with clinical outcome. In our study, high-avidity CTL lines could be expanded ex vivo from an antigen-primed animal using low peptide concentration, and intermediate peptide concentrations favored the generation of lower avidity CTL. Further increases in peptide concentration during culture inhibited the expansion of all peptide-specific CD8+ cells. In contrast, a single amino acid variant peptide efficiently generated functional CTL populations at high or low peptide concentration, which responded to wild-type epitope with the lowest average avidity seen in this study. We propose that for some peptides, the efficient generation of low-avidity CTL responses will be favored by stimulation with altered peptide rather than high concentrations of wild-type epitope. In addition, some variant peptides designed to have improved binding to major histocompatibility complex class I may reduce rather than enhance the functional avidity for the wild-type peptide of ex vivo-expanded CTL. These observations are relevant to in vitro expansion of CTL for immunotherapy and strategies to elicit regulatory or therapeutic immunity to neo-self-antigen when central tolerance has eliminated high-avidity, cognate T cells.

Key Words: CTL • variant peptides • T cell receptors • T cell growth • adoptive immunotherapy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cytotoxic T lymphocyte (CTL) response to antigenic peptides presented by major histocompatibility complex (MHC) class I is characterized by expansion of a heterogeneous mix of clones, each expressing a unique T cell receptor (TCR). For any given peptide/MHC (pMHC) complex, there generally exists a spectrum of TCRs (and therefore, cells) capable of binding pMHC with different affinities. In general, the affinity of the TCR for pMHC is low, and multiple ligand pairings at the cellular interface between CTL and antigen-presenting cells (APC) produce an immunological synapse that acts to concentrate TCR and pMHC for optimal interaction [1 ]. Once a stable synapse is formed, sustained TCR ligation activates signaling pathways that ultimately result in various effector functions [2 ]. Small changes in the concentration of TCR, pMHC, or both, in addition to TCR affinity, can dramatically alter the downstream signaling within the T cell [3 ]. Given that simultaneous measurement of multiple surface interactions on living cells is difficult, many studies titrate peptide ligand on the APC and measure the resulting CTL effector response to estimate the relative "functional" avidity of a CTL population for a pMHC complex [4 5 ]. High-avidity CTL are then defined as responding functionally to low concentrations of exogenous peptide, and conversely, low-avidity cells require relatively high concentrations of peptide to be activated.

In vitro culture conditions, based on peptide concentration, have been established, which selectively expand high- and low-avidity CTL from a mixed starting population for a range of peptides [4 6 7 ]. A key feature of ex vivo selection of antigen-primed T cells is that high-avidity CTL are not selected for expansion when presented with high peptide concentrations, and there is an outgrowth of lower avidity clones [8 ]. Conversely, low-avidity clones do not expand efficiently at low peptide concentration, and expansion of high-avidity clones is favored. In vivo, high-avidity CTL have been shown to be superior to low-avidity CTL in the clearance of viral infections and some tumors, and low-avidity populations are efficient in the clearance of tumors overexpressing self-proteins [7 9 10 11 12 ]. Despite the physiological importance of these populations, less is known about the selection of different T cell avidities in vivo, although several reports now suggest that primed CTL populations mature to a higher avidity phenotype during a typical immune response to infection [13 14 ]. The accumulation of higher avidity CTL has also been correlated with progression to autoimmune disease in animal models [15 ]. Little evidence to date suggests that a skewing toward low-avidity clones can occur during the natural immune response to infection. One study demonstrated that the immune response to recombinant vaccinia was composed mostly of low-avidity clones with high-avidity clones increasing at the acute stage of the infection [16 ].

In this study, we investigated the in vitro requirements for selective expansion of low- or high-avidity CTL populations directed against a murine CTL epitope of the human papillomavirus type 16 (HPV16)E7 protein as a model tumor antigen. We show that the lowest avidity, E7-specific CTL, is not efficiently expanded by high E7 peptide concentration in vitro. Instead, a variant E7 peptide, with a single amino acid change in the binding pocket at position 9, resulted in the expansion of low-avidity, wild-type, E7-specific CTL. Such information will enhance vaccine design or adoptive immunotherapy protocols, where CTL, of a particular avidity, are being targeted for expansion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Peptides, antibodies, and E7 wild-type (GF; RAHYNIVTF) peptide/H-2Db tetramer
The E7(GF) peptide and the variant E7 peptide (GV; RAHYNIVTV) were synthesized and purified as described previously [17 ]. TCRVß antibodies were a kind gift of Dr. Robert Tindle (Sir Albert Sakewski Virus Research Centre, Royal Children’s Hospital, Brisbane, Australia). Biotinylated, APC, or phycoerythrin (PE)-conjugated rat anti-mouse CD8{alpha} (clone 53-6.7) antibodies and streptavidin-PE were obtained from PharMingen (San Diego, CA). For the analysis of murine interferon-{gamma} (IFN-{gamma}) in culture supernatants, the capture antibody (clone R4-6A2) and the biotinylated detection antibody (clone XMG1.2) were also purchased from PharMingen. GF peptide/H-2Db tetramers labeled with PE or APC were obtained from the National Institutes of Health Tetramer Facility (AIDS Research and Reference Reagent Program, Bethesda, MD).

Mice and immunization
Adult C57Bl/6J mice (6–8 weeks) were obtained from the Animal Resource Centre (Perth, Australia) and were maintained at the Princess Alexandra Hospital Biological Research Facility by trained laboratory animal technicians. All animal experiments received ethical approval from the University of Queensland Animal Ethics Committee. C57BL/6J mice were immunized subcutaneously in the tail base with 50–100 µg nonamer peptide or HPV16E7-glutathione S-transferase protein mixed with 10 µg Quil A (Spikoside, Isotec AB, Sweden) adjuvant. Spleens were taken between days 6 and 21 following immunization.

Generation of CTL lines/clones and TCR-ß CDR3 sequencing
We have generated CTL lines at different peptide doses in at least three independent experiments. Single-cell suspensions were prepared from pooled spleens or the spleens of individual mice immunized with peptide. Cells were next treated with ammonium chloride lysis buffer to remove red blood cells and were then resuspended in complete tissue culture media [50% Eagle’s Ham’s amino acids/50% RPMI /10% fetal calf serum (FCS)/penicillin/streptomycin/glutamine/5x10–5 2-mercaptoethanol]. Cells were counted using a haemocytometer and trypan blue exclusion. Splenocytes from the peptide-immunized mice were plated into a 24-well plate at 7.5 million cells/well. Stimulator cells were harvested from the spleens of unimmunized mice in a similar manner. Stimulators at 10 million/mL were pulsed in tubes with varying concentrations of peptide for a minimum of 2 h at 37°C, followed by irradiation with 3000 rad of {gamma} radiation. Stimulators were washed with tissue-culture media before plating with the effectors in a 24-well plate. Stimulators were added to effectors at 3.5 million/well in addition to murine recombinant interleukin (mrIL)-2 (PharMingen) at 1 ng/mL (approximately 10 IU/mL). Cultures were placed at 37°C for 7 days before stimulating again. Subsequent rounds of stimulation involved the addition of 0.5 million harvested effectors to 5 million irradiated, peptide-pulsed splenocytes in a 24-well plate with the addition of mIL-2. In some experiments, CD8+ cells were depleted from the stimulator spleen population using magnetic anti-CD8 Dynabeads, according to the manufacturer’s instructions (Dynal Biotech, Oslo, Norway). Depletion of CD8 cells was greater than 95% in these experiments.

Clones were derived from established, E7-specific CTL lines having undergone at least six stimulations in vitro. Cloning was performed using limiting dilution in 96-well round-bottom plates. Irradiated, peptide-pulsed splenocytes were added to each well (5 million cells) along with effector CTL at 10, 5, 2, 1, and 0.1 cells per well and mIL-2. The identification of clonal T cell cultures was determined microscopically and was statistically based on a Poisson distribution for the frequency of "growing" wells. Clones were gradually expanded to 24-well plates by weekly stimulation cycles.

RNA was extracted from cells of several individual CTL clones using Trizol reagent (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. cDNA was then generated using a Gene Amp RNA polymerase chain reaction (PCR) kit (Perkin Elmer, Wellesley, MA) according to the manufacturer’s instructions using random hexamer or oligo dT primers. A fragment of the TCR-ß chain was amplified using a 5' TCRVß-degenerate primer and a 3' TCRCß primer (5' CAGCTCAGCTCCACGTGG 3') as described previously [18 ]. The entire PCR product was then purified using a Wizard PCR prep kit (Promega, Sydney, Australia) and precipitated with isopropanol. Sequencing was performed using the 3' TCRCß primer and Big Dye terminator mix (ABI, Foster City, CA) and was analyzed on an ABI automated sequencer.

Flow cytometry
CTL lines and clones were stained with a panel of TCRVß antibodies (Vß 2-14+17; gift of Dr. Robert Tindle). CTL were placed in tubes at 1–5 x 105 cells/tube in a 100-µL vol to which 10 µL each monoclonal antibody supernatant was added. Cells were incubated at 4°C for 1 h before washing three times in phosphate-buffered saline (PBS)/1% FCS. Fluorescein isothiocyanate-conjugated anti-rat immunoglobulin (Ig), anti-hamster Ig, or anti-mouse Ig conjugates were then added at ~1 µg/tube in a 100-µL vol. Cells were again incubated at 4°C for 1 h before washing to remove free antibody and fixing in 2% paraformaldehyde. Fluorescence was examined using a FACScan flow cytometer and CellQuest software (BD Biosciences, San Diego, CA).

For tetramer staining, 1 x 105 CTL were simultaneously incubated with 1 µg anti-CD8{alpha} PE- or APC-labeled antibody and 50 nM E7(GF)/H-2Db tetramer conjugated with PE or APC fluorochrome. Cells were labeled for 1 h at 4°C before washing and fixing in 2% paraformaldehyde. Fluorescence was analyzed in a FACScan using CellQuest software.

Propidium iodide (PI; Sigma-Aldrich, Sydney, Australia) staining was performed on 5 x 105 cells by resuspending the cells in a solution of 4 µg/mL PI in PBS/5% FCS. After 20 min, cells were analyzed on the FACScan using CellQuest software.

Cytotoxicity assay
The cytotoxicity assay was performed as described previously [19 ]. Briefly, 5 x 105 EL-4 targets (ATCC TIB-39) were placed in a volume of 100 µL in a 15-mL tube. Peptide was added at different concentrations at the same time as 100 µCi 51Cr. The tube was incubated for 2 h at 37°C before washing to remove soluble 51Cr and peptide from the cells. Targets were plated into a round-bottom 96-well plate at 3000 cells/well, and effector cells were then added to the plate at the indicated effector:target (E:T) ratios. Plates were centrifuged at 200 g for 2 min to pellet the cells, and then the plate was incubated at 37°C for 4–5 h. Following this incubation, the plate was again centrifuged at 200 g for 5 min, and then 25 µL supernatant was transferred to a Lumaplate (Packard, Australia) containing a solid scintillant. The Lumaplate was placed overnight in a drying oven before scintillation counts were determined on a Packard TopCount NXTTM. The percent lysis was calculated from the means of triplicate wells as follows: (mean experimental 51Cr release) – (mean spontaneous 51Cr release) x 100/(mean maximum 51Cr release) – (mean spontaneous 51Cr release).

Spontaneous release was determined from wells containing labeled cells and media, and maximum release was determined from wells containing 5% sodium dodecyl sulfate and labeled cells. Results were recorded as percent specific lysis following subtraction of experimental lysis values for effectors incubated with targets without peptide. Lysis of EL-4 targets in the absence of peptide was less than 6% in all assays.

Proliferation, IFN-{gamma}, enzyme-linked immunosorbent assay (ELISA), and IFN-{gamma} enzyme-linked immunospot (ELISPOT)
C57Bl/6 spleen cells were harvested as described earlier and incubated with varying concentrations of peptide for 2–3 h at 37°C. After washing to remove free peptide, cells were irradiated (3000 rad) and plated into 96-well round-bottom plates at 3 x 105 cells/well. CTL lines (>day 7 poststimulation) were harvested from 24-well plates, washed, and plated in the round-bottom wells (with the stimulators) at 5 x 104 cells/well. Plates were incubated at 37°C for 48 h before removing 100 µL supernatant for the detection of secreted IFN-{gamma}. Supernatants were stored frozen (–20°C) before assay. IFN-{gamma} was detected using a capture ELISA according to the manufacturer’s instructions (PharMingen).

For proliferation assays, 1 µCi tritiated thymidine, dissolved in complete media, was replaced in the wells described above at 48 h, and the plate was incubated at 37°C. After overnight incubation, cells were harvested, and incorporation of [3H]thymide was measured. Stimulation index (SI) was calculated as: [counts per minute (cpm) for cells and peptide]/(cpm for cells without peptide). The % maximal SI was calculated as (SI for experimental value) x 100/(SI for highest experimental value).

The IFN-{gamma} ELISPOT was performed as described previously using in vitro-cultured effector cells and 1 x 106 irradiated spleen cells as filler cells [20 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Evolution of HPV16E7 peptide-specific CTL cultures using different peptide concentrations
To investigate the role of peptide concentration as a determinant of the in vitro expansion of E7-specific T cells of a particular functional avidity, spleen cells from E7 protein-immunized mice were incubated with target splenocytes pulsed with 10 µM or 0.01 µM of the dominant H-2Db-restricted CD8 epitope of E7, RAHYNIVTF ("GF peptide") in the presence of exogenous IL-2. Our consistent finding was that splenocytes exposed long-term to 10-µM GF peptide were unable to develop functional CTL responses to the GF peptide (Fig. 1A ). This contrasted with splenocytes exposed to 0.01-µM GF peptide, where functional responses to the peptide developed after a single round of in vitro stimulation, and CTL lines were maintained long-term (Fig. 1A and 1C) .



View larger version (30K):
[in this window]
[in a new window]
  		Figure 1. High GF peptide concentration does not favor long-term expansion of E7(GF)-specific CTL. (A) Long-term CTL cultures generated by weekly stimulation for 7 weeks with the 10-µM GF peptide (10 µM GF culture) or the 0.01-µM GF peptide (0.01 µM GF culture) were tested for cytotoxicity against EL-4 targets untreated (NO PEPT.) or pulsed with GF peptide (GF PEPT.) or ovalbumin (OVA) peptide (SIINF PEPT.). The assay was conducted over 4–5 h at an E:T ratio of 10. Error bars represent the standard deviation of triplicate cultures. (B) Spleen cells from E7-immunized mice were cultured with rIL-2, and irradiated spleen cells were pulsed with 10-µM or 0.01-µM E7 peptide. At day 6 poststimulation, a portion of the cultures was stained with anti-mouse CD8{alpha} antibody and 50 nM GF peptide/H-2Db tetramer at 4°C for at least 1 h. Data are gated on live cells based on forward- and side-scatter properties, and percentages indicate tetramer-positive cells within the CD8+ population. Stimulation of E7-specific CTL with peptide-pulsed stimulators and IL-2 occurred every 7 days, and the development of CTL was monitored by tetramer staining (on day 6 or 7 poststimulation) over 4 weeks. (C) Spleen cells from GF001-immunized mice were stimulated with irradiated splenocytes pulsed with 10-µM or 0.01-µM GF peptide and mIL-2 for 1 week. Cells were then harvested and placed in an overnight IFN-{gamma} ELISPOT assay (SPOTS). Error bars represent the standard deviation of replicate cultures. All experiments are representative of at least two independent experiments.

 
Given previous data suggesting that high peptide concentration is a major determinant for the development of low-avidity CTL cultures, we decided to explore the generation of low-avidity CTL in the HPV16E7 peptide system. To determine the stage at which high peptide conditions inhibit the production of functional GF peptide-specific CTL, we assessed the evolution of CTL lines exposed to different GF peptide concentrations in vitro by GF peptide/H-2Db tetramer staining. Tetramer-positive, CD8+ T cells were undetectable (<1%) amongst splenocytes taken directly ex vivo from an animal primed by a single immunization with E7 protein (data not shown). After one round of in vitro stimulation, minimal GF peptide-specific CTL numbers could be detected with tetramers, although tetramer-positive cells were marginally increased in the cultures grown on 0.01-µM GF peptide. After a second stimulation, the difference in the number of tetramer-binding cells between low and high peptide concentrations was apparent and became more obvious following the third and fourth stimulations (Fig. 1B) . Expression of CD3 on cells from 0.01-µM GF culture or 10-µM GF culture was not significantly different when measured at week 2 (0.01 µM culture=115 fluorescence units; 10 µM culture=137 fluorescence units). The early inhibition of E7-specific CTL expansion by high GF peptide concentrations was supported by functional data obtained using an IFN-{gamma} ELISPOT after a single round of in vitro stimulation in an independent experiment (Fig. 1C) . In this experiment, minimal numbers of IFN-{gamma}-secreting cells were observed from cultures grown on 10-µM GF peptide in comparison with the higher numbers seen with 0.01-µM GF peptide. Consequently, the functional data were consistent with the tetramer staining and suggested an immediate inhibition of E7-specific CTL expansion under high peptide conditions in vitro, which cannot be overcome by continued long-term culture of the cells.

One possibility was that high concentrations of GF peptide, soluble or cell-bound, are generally toxic to the development of CTL cultures, despite extensive washing of the peptide-pulsed stimulator cells. This possibility was investigated by adding 10-µM GF peptide (H-2Db-restricted) to a standard set of culture conditions for generating OVA-specific CTL lines from OVA-immunized mice. Splenocytes, cultured with 0.01-µM OVA peptide-pulsed stimulators in the presence or absence of a high concentration E7 peptide, were similar in the ability to generate IFN-{gamma}-secreting, OVA-specific cells (Fig. 2A ). This suggested that the 10-µM GF peptide was not simply toxic but instead, was acting directly on the GF peptide-specific population.



View larger version (36K):
[in this window]
[in a new window]
  		Figure 2. High concentrations of GF peptide lead to GF-specific CTL death but do not affect the development of CTL of unrelated specificity. (A) Splenocytes, from mice immunized with OVA peptide (SIINF), were cultured with irradiated splenocytes pulsed with 0.01 µM SIINF in the presence or absence of the 10-µM GF peptide and mIL-2 for 1 week. Cells were harvested and stimulated with SIINFEKL peptide overnight in an IFN-{gamma} ELISPOT assay (SPOTS). Error bars represent the standard deviation of replicate cultures. (B) Subcultures of a long-term, CD8+, E7-specific CTL (>95% E7 tetramer-positive) line grown routinely on the 0.01-µM GF peptide were exposed to CD8-depleted, irradiated spleen cells pulsed with the 10-µM GF peptide (10 µM GF Stimulators) or the 0.01-µM GF peptide (0.01 µM GF Stimulators). After 48 h, both subcultures were stained with APC-conjugated antibody against CD8{alpha} (CD8-APC) and PI (FL2-PI). The double-hatched region represents 6000 gated CD8+ cells (i.e., excluding stimulator cells, which are CD8–), which were analyzed for PI uptake in the lower panels. The percentage of gated cells that fails to exclude PI is indicated. (C) Splenocytes, derived from HPV16E7 peptide/protein-immunized mice, were stimulated weekly for 4 weeks on the 10-µM GF peptide (10 µM GF CTL) or the 0.01-µM GF peptide (0.01 µM GF CTL) before being given a single stimulation cycle using the 0.01-µM GF peptide (0.01 µM GF Stimulators) or the 10-µM GF peptide (10 µM GF Stimulators). Cells from these four cultures were then stained with anti-mouse CD8{alpha} antibody and GF peptide/H-2Db tetramer at day 6 poststimulation. Live cells were gated for analysis, and percentages above each plot represent the proportion of gated cells positive for CD8{alpha} and tetramer. All experiments are representative of at least two independent experiments.

 
To determine if high GF peptide concentrations induced cell death in the E7-specific CTL, subcultures of a high-avidity, E7-specific CTL line were exposed to low (0.01 µM)- or high (10 µM)-dose GF peptide pulsed onto CD8-depleted, irradiated spleen cells. Depletion of CD8+ cells from the stimulator population enabled the cells of the CTL line to be discriminated via CD8 staining. E7-specific CTL exposed to high GF peptide concentration for 48 h had an increased number of PI-staining cells (dead cells) amongst gated CD8+ populations compared with exposure of the same CTL population to low peptide concentration (region R2; Fig. 2B ). In addition, the proportion of CD8{alpha}hi cells was markedly decreased under the high GF peptide conditions (region R1; Fig. 2B ). This suggests that at least a substantial proportion of high-avidity, E7-specific CTL undergo cell death when exposed to high GF peptide concentrations and that CD8 down-regulation may be associated with that process.

After CTL exposure to high GF peptide concentration, we noted that small numbers of live cells persisted in the culture. It was possible that small numbers of E7-specific CTL were chronically suppressed by high E7 peptide concentration and that a switch in culture conditions to low-dose peptide might expand this population. To determine whether short-term E7-specific CTL lines (fourth stimulation), developed in high peptide concentration, were able to expand in a lower GF peptide concentration, subcultures of cell lines routinely stimulated by low or high GF peptide were switched to high or low concentrations of GF peptide for a single stimulation cycle (Fig. 2C) . Subcultures initiated and maintained on high E7 peptide concentration showed similar numbers of tetramer-positive CTL 1 week after changing to low-dose peptide. This suggests that provision of low-dose peptide does not "reveal" a large population of tetramer+ CD8+ cells whose tetramer staining or expansion may have been compromised by continuous growth in high concentrations of GF peptide. CTL cultures initiated and maintained in low GF peptide concentrations, when stimulated with high E7 peptide and gated on live cells, demonstrated decreased staining intensity of the tetramer-positive population (Fig. 2C) , suggesting that short-term exposure to high E7 peptide concentrations leads to down-regulation of TCR on the viable, high-avidity, E7-specific CTL population. A larger fraction of CD8– tetramer– cells was also observed under these conditions.

Thus, high E7 peptide concentration leads to deletion of high-avidity CTL populations, as previously reported in several other peptide systems [4 6 7 ] but does not simultaneously favor the expansion of low-avidity CTL clones in vitro.

Intermediate peptide concentrations favor a shift toward lower functional avidity
It was possible that intermediate E7 peptide concentrations between 10 µM and 0.01 µM might still promote the growth of low-avidity CTL populations. Long-term, E7-specific CTL could be generated using peptide concentrations ranging from 0.1-µM to 0.001-µM peptide (Fig. 3 ). CTL cultures grown over this two-log range in E7 peptide concentration were not grossly different in their functional avidity based on lytic assays or proliferation (Fig. 3A and 3D) . However, 0.1-µM and 0.001-µM E7-specific CTL cultures varied in their ability to secrete IFN-{gamma} in response to titrated concentrations of cognate peptide, suggesting that this assay was able to discriminate a difference in avidity, where lysis of peptide-pulsed targets did not (Fig. 3B) . Previous studies have shown a key role for CD8 in enhancing TCR/pMHC interaction and demonstrated that CD8 dependence inversely correlates with TCR affinity/avidity in lytic assays [8 21 ]. We hypothesized that blocking CD8 interactions in a lytic assay might reveal differences in the functional avidity of the E7-specific CTL lines, consistent with the IFN-{gamma} assay. In the presence of a blocking CD8{alpha} antibody, but not otherwise, cell lines grown on a 0.1-µM E7 peptide required higher peptide concentrations on target cells to achieve an equivalent level of lysis than lines grown on 0.001-µM peptide, despite similar expression levels of CD8{alpha} protein (0.1 µM line=478 fluorescence units; 0.001 µM line=470 fluorescence units; Fig. 3C ). We therefore suggest that lower avidity, E7-specific CTL do exist and that they can be expanded from in vivo-primed cells with intermediate concentrations of the E7 peptide.



View larger version (38K):
[in this window]
[in a new window]
  		Figure 3. Intermediate and low concentrations of GF peptide generate CTL with differences in functional avidity. CTL cultures grown on three different concentrations of GF peptide (0.1, 0.01, or 0.001 µM) were analyzed for lytic activity (A), IFN-{gamma} production (B), lytic activity in the presence of blocking anti-CD8{alpha} antibody (C), and proliferation (D). Error bars represent the standard deviation of triplicate wells. These experiments are representative of at least two independent experiments. ABS, absorbance at 490 nm.

 
Given the expansion of lower avidity CTL at the 0.1-µM E7 peptide, it was unusual that higher peptide concentrations completely inhibited the CTL response, which to defined antigens, can be dominated by the expansion of a limited number of T cell clones [22 23 ]. Consequently, the inability to generate E7-specific CTL at the 10-µM E7 peptide might reflect a limited number of clones capable of responding to the E7 peptide. To examine the diversity of the E7-specific T cell repertoire, several T cell clones were generated by limiting dilution from independently derived, E7-specific CTL cultures of differing functional avidity (grown on 0.1–0.001 µM E7 peptide as described above) and were analyzed for their TCRVß use. Analysis of these CTL clones and lines revealed use of at least five different TCRVß chains [0.1 µM CTL (five clones)=TCRVß8.3 or 9; 0.01 µM CTL (12 clones)=TCRVß12 or 8.1; 0.001 µM CTL (two clones)=TCRVß6] and at least two different TCRVß CDR3 amino acid sequences (0.01 µM Vß12 clone=LGDNYAEQF; 0.001 µM Vß6 clone=IGFANTEVF). Together, these data suggest that the GF peptide-specific CTL response was not monomorphic.

Consequently, the inability to generate E7-specific CTL at 10-µM E7 peptide is more likely to be a property of this peptide and its binding interaction with MHC and/or TCR rather than a deficiency in the available T cell repertoire.

Alteration of the E7 peptide at the position 9 anchor residue allows expansion of the lowest avidity CTL to the wild-type E7 peptide
We next investigated whether the restriction on the generation of CTL at 10-µM E7 peptide was a property of the peptide. A variant E7 peptide with a single substitution at position nine demonstrates enhanced binding to H-2Db and generates CTL cross-reactive with the wild-type E7 peptide [24 ]. Using the variant peptide (designated GV), we investigated the expansion of high- and low-avidity CTL populations in this peptide system. GV peptide-specific CD8 T cell populations, identified using the cross-reactive GF peptide/H-2Db tetramer (Fig. 4A ) or IFN-{gamma} ELISPOT (Fig. 4B) , were rapidly enriched after a single stimulation with 10-µM or 0.01-µM GV peptide (week 1) and continued to enrich through to week 3. This contrasts with the expansion of GF peptide-specific CTL populations, where 10-µM peptide failed to produce IFN-{gamma}-secreting cells (Fig. 4B) or GF peptide/H-2Db tetramer-staining cells (Fig. 1B) over the same time-period. In addition, the expanded GV-specific CTL populations took three rounds of in vitro peptide stimulation to show a separation in relative functional avidity of the population (Fig. 4B ; lower left panel). This confirmed that a single amino acid change in the wild-type GF peptide produces a peptide (GV) capable of efficiently expanding functional GV-specific CTL at 10-µM peptide concentration.



View larger version (34K):
[in this window]
[in a new window]
  		Figure 4. A variant E7 peptide (GV) rapidly generates high- and low-avidity, GV-specific CD8 T cells in vitro. (A) Splenocytes from GV peptide-immunized mice were stimulated with a high GV peptide concentration (10 µM GV culture; left panels) or a low peptide concentration (0.01 µM GV culture; right panels), pulsed onto irradiated splenocytes. The development of peptide-specific CTL cultures was monitored after 1 week (upper panels) or 3 weeks (lower panels) using anti-CD8{alpha} antibody in combination with GF peptide/H-2Db tetramers in flow cytometry. Plots are gated on live cells as determined by forward- and side-scatter, and a representative experiment of two experiments is shown. (B) Splenocytes from GV (left panels)- or GF (right panels)-immunized mice were stimulated with a high (10 µM cultures) or low concentration (0.01 µM cultures) of their cognate peptide pulsed onto irradiated splenocytes each week for a period of 1 week (upper panels) or 3 weeks (lower panels). Functional avidity of the cultured cells was tested using the cognate peptide in an IFN-{gamma} ELISPOT assay (SPOTS). The error bars represent the standard deviation of replicate cultures. All experiments are representative of at least two independent experiments.

 
Given that the GV peptide induces CTL, which cross-react with the wild-type GF peptide, we next investigated the functional avidity of GV-specific CTL populations when assayed on the GF peptide. It is surprising that GV-specific CTL lines, when assayed against the cross-reactive, wild-type GF peptide, now revealed the lowest avidity CTL populations for GF that we have observed in this study (Fig. 5A ). Both GV CTL lines fail to respond below 10–2-µM GF peptide, whereas all GF CTL lines generated throughout this study titrate to at least 10–5-µM GF peptide. The GV peptide was required as the immunogen and stimulating peptide in vitro to generate these low-avidity, GF-specific CTL, given that cells from GF peptide-immunized animals stimulated with GV peptide in vitro had a higher average avidity (Fig. 5A) . Although, both GV-specific CTL cultures had low avidity for the GF peptide, these CTL demonstrated differences in functional avidity when tested on their cognate peptide (Fig. 5B) , consistent with earlier results (Fig. 4B) . In addition, CTL cultures derived from mice immunized with GF peptide and stimulated with GV peptide in vitro showed a minor difference in functional avidity when tested on the GV peptide. This suggests that the GV peptide readout has a greater propensity to reveal differences in functional avidity.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 5. The variant E7 peptide elicits high- and low-avidity CTL for the variant E7(GV) and low-avidity CTL for the wild-type E7(GF) peptide. C57Bl/6 mice were immunized with the variant E7 peptide (GV Imm.) or the wild-type E7 peptide (GF Imm.), and spleen cells were restimulated in vitro with the 10-µM (10 µM GV) or the 0.01-µM (0.01 µM GV) peptide to establish long-term CTL lines. These lines were then tested for cytotoxicity using EL-4 cells pulsed with varying concentrations of the wild-type E7(GF) peptide (A) or the variant E7(GV) peptide (B) at E:T ratios of 20 and 15, respectively. These experiments represent one of two similar experiments, and error bars represent the standard deviation of triplicate wells.

 
Finally, the data suggest that low-avidity, E7-specific CTL repertoires exist within mice but that the altered peptide, when used as the immunogen and stimulant in vitro, is more efficient than high concentrations of the wild-type peptide in promoting their expansion.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Using a model, immunodominant, H-2Db-restricted CTL epitope of the HPV16E7 protein in C57BL/6 mice, we observed that long-term CTL lines can be generated ex vivo from primed animals if the concentration of peptide pulsed onto stimulator cells was between 0.1 and 0.001 µM. Higher peptide concentrations inhibited the expansion of functional, E7 tetramer-binding CTL, inducing cell death in a substantial fraction of the cells. The continued presence of tetramer-negative CD8 cells in 10-µM peptide conditions probably reflects the general survival of CD8 cells in culture medium containing exogenous IL-2 rather than an enrichment of E7-specific cells (Fig. 1B) .

The simplest model for our data would suggest that cells with a full range of avidities for E7 peptide (GF) can be expanded between 0.1- and 0.001-µM peptide and that higher peptide concentrations deliver an inhibitory/death signal to all E7-specific CTL. We believed that such a narrow window of responsive CTL was unusual at these relatively low-peptide concentrations. Several studies have reported selective growth of low-avidity CTL lines using lymphocytes from primed animals exposed in vitro to high peptide concentrations (up to 100 µM) as a result of simultaneous deletion of high-avidity CTL present in the same culture [4 6 ]. The deletion of high-avidity CTL at high peptide concentration would be consistent with our result and other studies, but the failure of high-dose cognate peptide (10 µM) to expand low-avidity CTL would not be generally predicted from the literature [8 25 ]. In one study where CTL of lower avidity could not be produced readily following paramyxovirus infection of mice and peptide restimulation, the basis of this observation was not investigated [26 ].

Failure of 10-µM peptide to support the growth of low-avidity CTL could result from a deficiency in the available T cell repertoire or be a binding property of the peptide. We first chose to examine whether the absence of an E7-specific CTL response was indicative of a limited TCR repertoire for pMHC, as has been described for the response to superantigens and some peptides [22 27 28 ]. From within the E7-specific CTL cultures grown at low or intermediate peptide concentrations (0.1–0.001 µM), we generated clones using multiple TCRVß proteins/CDR3 sequences and demonstrated differences in functional avidity, suggesting that the available T cell repertoire is not limiting.

We next hypothesized that failure to generate E7-specific CTL at 10 µM might be a property of this particular peptide. Previous reports examining the generation of low-avidity responses to self-antigens have suggested that altered peptide ligands were more efficient than wild-type ligands in generating low-avidity T cell responses [29 30 ]. The TCR and MHC-binding residues in the E7 peptide/H-2Db complex have not been fully characterized. However, based on the binding motifs for H-2Db, one report has identified an E7 peptide with a single amino acid substitution at position 9, which was reported to enhance MHC binding and maintain T cell cross-reactivity with the wild-type E7 peptide [24 ]. Perhaps surprisingly, 10 µM (and 0.01 µM) of the variant E7 peptide (designated GV peptide) gave rise to GV-specific CTL lines ex vivo following priming and restimulation with this variant E7 peptide. This suggests that the inability to generate CTL against the wild-type E7 peptide (designated GF peptide) with high concentrations of GF peptide is not a product of inappropriate culture conditions and instead, is a structural property of the GF peptide. It is important that when the GV-specific CTL cultures were tested against the GF peptide, CTL populations with much lower average avidity than seen using the 0.1-µM GF peptide were observed. This confirmed that GV peptide immunization and in vitro stimulation were more effective than a high-concentration GF peptide in inducing the lowest avidity populations reactive with the GF peptide. This would be counterintuitive to the belief that peptides with improved MHC class I binding might be better stimulators of immune responses to the wild-type peptide.

Given that low-avidity, GF-specific CTL were generated at both concentrations of GV peptide (10 µM and 0.1 µM), it is unlikely that GV pMHC density is completely responsible for the expansion of these low-avidity, GF-specific cells. Instead, the expansion of low-avidity, GF-specific CTL may result from the increased stability of the GV peptide/H-2Db complex, such that the half-life of the pMHC complex rather than the density of pMHC selects the GF-specific CTL, which are expanded. Alternatively, it is possible that despite changing a MHC anchor residue, the variant pMHC presents an altered surface structure to responding T cells when compared with wild-type pMHC. This possibility is supported by a recent study of substituted peptides at MHC anchor residues in which the TCR contact residues (located at the center of the peptide) changed conformation, thereby altering the interaction with the TCR [31 ]. Low-avidity, GF-specific CTL might then be capable of being selectively expanded on this structure, an event that cannot be achieved by high concentrations of wild-type peptide. Determining the mechanism by which the GV peptide favors the development of low-avidity, GF-specific CTL would require structure-function studies involving a large panel of variant peptides based on the GF peptide sequence. It is interesting that the variant pMHC structure is also poor at inhibiting E7-specific CTL expansion, as GF peptide immunization followed by GV peptide stimulation allows development of GF-specific CTL, independent of GV concentration (Fig. 5A) . Consequently, pMHC density is not the sole determinant for the selection of CTL populations with different average avidities. Our data suggest a role for TCR interaction with individual pMHC complexes as a second selection pressure in determining the final avidity of a CTL population.

A major goal of immunotherapy is to generate CTL targeted to peptide derived from self-antigen to regulate existing, autoimmune responses or to eliminate tumors overexpressing self-antigen. As self-antigen generally deletes high-affinity CTL clones through positive and negative selection in the thymus, the repertoire of available, specific T cells in the periphery is restricted to those of lower affinity. Understanding the features of a peptide that enhance the expansion of low-avidity CTL will therefore be important for future vaccine design and adoptive immunotherapy protocols. Conversely, peptides that are designed to bind more stably to MHC than the wild-type peptide may not always enhance the quality of the CTL response.


   ACKNOWLEDGEMENTS
 
This work was supported by the Australian National Health and Medical Research Council (Grant No. 142 952), the University of Queensland Mayne Bequest Fund, the Lions Medical Research Foundation, and the Princess Alexandra Hospital Research Foundation. We acknowledge the technical assistance of Rachel deKluyver and Richard Linedale and the provision of reagents by Prof. Bob Tindle.

Received January 16, 2004; revised May 13, 2004; accepted June 3, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bromley, S. K., Burack, W. R., Johnson, K. G., Somersalo, K., Sims, T. N., Sumen, C., Davis, M. M., Shaw, A. S., Allen, P. M., Dustin, M. L. (2001) The immunological synapse Annu. Rev. Immunol. 19,375-396[CrossRef][Medline]
  2. Lanzavecchia, A., Sallusto, F. (2001) Antigen decoding by T lymphocytes: from synapses to fate determination Nat. Immunol. 2,487-492[CrossRef][Medline]
  3. Grakoui, A., Bromley, S. K., Sumen, C., Davis, M. M., Shaw, A. S., Allen, P. M., Dustin, M. L. (1999) The immunological synapse: a molecular machine controlling T cell activation Science 285,221-227[Abstract/Free Full Text]
  4. Alexander-Miller, M. A., Leggatt, G. R., Berzofsky, J. A. (1996) Selective expansion of high- or low-avidity cytotoxic T lymphocytes and efficacy for adoptive immunotherapy Proc. Natl. Acad. Sci. USA 93,4102-4107[Abstract/Free Full Text]
  5. Dutoit, V., Rubio-Godoy, V., Doucey, M. A., Batard, P., Lienard, D., Rimoldi, D., Speiser, D., Guillaume, P., Cerottini, J. C., Romero, P., Valmori, D. (2002) Functional avidity of tumor antigen-specific CTL recognition directly correlates with the stability of MHC/peptide multimer binding to TCR J. Immunol. 168,1167-1171[Abstract/Free Full Text]
  6. Zeh, H. J., III, Perry-Lalley, D., Dudley, M. E., Rosenberg, S. A., Yang, J. C. (1999) High avidity CTLs for two self-antigens demonstrate superior in vitro and in vivo antitumor efficacy J. Immunol. 162,989-994[Abstract/Free Full Text]
  7. Gallimore, A., Dumrese, T., Hengartner, H., Zinkernagel, R. M., Rammensee, H. G. (1998) Protective immunity does not correlate with the hierarchy of virus-specific cytotoxic T cell responses to naturally processed peptides J. Exp. Med. 187,1647-1657[Abstract/Free Full Text]
  8. Alexander-Miller, M. A., Leggatt, G. R., Sarin, A., Berzofsky, J. A. (1996) Role of antigen, CD8, and cytotoxic T lymphocyte (CTL) avidity in high dose antigen induction of apoptosis of effector CTL J. Exp. Med. 184,485-492[Abstract/Free Full Text]
  9. Derby, M., Alexander-Miller, M., Tse, R., Berzofsky, J. (2001) High-avidity CTL exploit two complementary mechanisms to provide better protection against viral infection than low-avidity CTL J. Immunol. 166,1690-1697[Abstract/Free Full Text]
  10. Morgan, D. J., Kreuwel, H. T., Fleck, S., Levitsky, H. I., Pardoll, D. M., Sherman, L. A. (1998) Activation of low avidity CTL specific for a self epitope results in tumor rejection but not autoimmunity J. Immunol. 160,643-651[Abstract/Free Full Text]
  11. Sedlik, C., Dadaglio, G., Saron, M. F., Deriaud, E., Rojas, M., Casal, S. I., Leclerc, C. (2000) In vivo induction of a high-avidity, high-frequency cytotoxic T-lymphocyte response is associated with antiviral protective immunity J. Virol. 74,5769-5775[Abstract/Free Full Text]
  12. Yee, C., Savage, P. A., Lee, P. P., Davis, M. M., Greenberg, P. D. (1999) Isolation of high avidity melanoma-reactive CTL from heterogeneous populations using peptide-MHC tetramers J. Immunol. 162,2227-2234[Abstract/Free Full Text]
  13. Busch, D. H., Pamer, E. G. (1999) T cell affinity maturation by selective expansion during infection J. Exp. Med. 189,701-710[Abstract/Free Full Text]
  14. Slifka, M. K., Whitton, J. L. (2001) Functional avidity maturation of CD8(+) T cells without selection of higher affinity TCR Nat. Immunol. 2,711-717[CrossRef][Medline]
  15. Amrani, A., Verdaguer, J., Serra, P., Tafuro, S., Tan, R., Santamaria, P. (2000) Progression of autoimmune diabetes driven by avidity maturation of a T-cell population Nature 406,739-742[CrossRef][Medline]
  16. Alexander-Miller, M. A. (2000) Differential expansion and survival of high and low avidity cytotoxic T cell populations during the immune response to a viral infection Cell. Immunol. 201,58-62[CrossRef][Medline]
  17. Fernando, G. J., Tindle, R. W., Frazer, I. H. (1995) T-helper epitopes of the E7 transforming protein of cervical cancer associated human papillomavirus type 18 (HPV18) Virus Res 36,1-13[CrossRef][Medline]
  18. Fox, C. J., Danska, J. S. (2003) Molecular analysis of mouse T cell receptor expression using PCr Colligan, J. E. Druisbeek, A. M. Margulies, D. H. Shevach, E. M. Strober, W. eds. Current Protocols in Immunology John Wiley & Sons
  19. Leggatt, G. R., Hosmalin, A., Pendleton, C. D., Kumar, A., Hoffman, S., Berzofsky, J. A. (1998) The importance of pairwise interactions between peptide residues in the delineation of TCR specificity J. Immunol. 161,4728-4735[Abstract/Free Full Text]
  20. Gao, F. G., Khammanivong, V., Liu, W. J., Leggatt, G. R., Frazer, I. H., Fernando, G. J. (2002) Antigen-specific CD4+ T-cell help is required to activate a memory CD8+ T cell to a fully functional tumor killer cell Cancer Res 62,6438-6441[Abstract/Free Full Text]
  21. Alexander, M. A., Damico, C. A., Wieties, K. M., Hansen, T. H., Connolly, J. M. (1991) Correlation between CD8 dependency and determinant density using peptide-induced, Ld-restricted cytotoxic T lymphocytes J. Exp. Med. 173,849-858[Abstract/Free Full Text]
  22. Lawson, T. M., Man, S., Williams, S., Boon, A. C., Zambon, M., Borysiewicz, L. K. (2001) Influenza A antigen exposure selects dominant Vß17+ TCR in human CD8+ cytotoxic T cell responses Int. Immunol. 13,1373-1381[Abstract/Free Full Text]
  23. Kalams, S. A., Johnson, R. P., Trocha, A. K., Dynan, M. J., Ngo, H. S., D’Aquila, R. T., Kurnick, J. T., Walker, B. D. (1994) Longitudinal analysis of T cell receptor (TCR) gene usage by human immunodeficiency virus 1 envelope-specific cytotoxic T lymphocyte clones reveals a limited TCR repertoire J. Exp. Med. 179,1261-1271[Abstract/Free Full Text]
  24. Vierboom, M. P., Feltkamp, M. C., Neisig, A., Drijfhout, J. W., ter Schegget, J., Neefjes, J. J., Melief, C. J., Kast, W. M. (1998) Peptide vaccination with an anchor-replaced CTL epitope protects against human papillomavirus type 16-induced tumors expressing the wild- type epitope J. Immunother. 21,399-408
  25. Critchfield, J. M., Racke, M. K., Zuniga-Pflucker, J. C., Cannella, B., Raine, C. S., Goverman, J., Lenardo, M. J. (1994) T cell deletion in high antigen dose therapy of autoimmune encephalomyelitis Science 263,1139-1143[Abstract/Free Full Text]
  26. Gray, P. M., Parks, G. D., Alexander-Miller, M. A. (2001) A novel CD8-independent high-avidity cytotoxic T-lymphocyte response directed against an epitope in the phosphoprotein of the paramyxovirus simian virus 5 J. Virol. 75,10065-10072[Abstract/Free Full Text]
  27. Abe, R., Vacchio, M. S., Fox, B., Hodes, R. J. (1988) Preferential expression of the T-cell receptor V ß 3 gene by Mlsc reactive T cells Nature 335,827-830[CrossRef][Medline]
  28. Marrack, P., Kushnir, E., Kappler, J. (1991) A maternally inherited superantigen encoded by a mammary tumour virus Nature 349,524-526[CrossRef][Medline]
  29. de Visser, K. E., Cordaro, T. A., Kessels, H. W., Tirion, F. H., Schumacher, T. N., Kruisbeek, A. M. (2001) Low-avidity self-specific T cells display a pronounced expansion defect that can be overcome by altered peptide ligands J. Immunol. 167,3818-3828[Abstract/Free Full Text]
  30. Anderton, S. M., Radu, C. G., Lowrey, P. A., Ward, E. S., Wraith, D. C. (2001) Negative selection during the peripheral immune response to antigen J. Exp. Med. 193,1-11
  31. Sharma, A. K., Kuhns, J. J., Yan, S., Friedline, R. H., Long, B., Tisch, R., Collins, E. J. (2001) Class I major histocompatibility complex anchor substitutions alter the conformation of T cell receptor contacts J. Biol. Chem. 276,21443-21449[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0104026v1
76/4/787    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Leggatt, G. R.
Right arrow Articles by Frazer, I. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Leggatt, G. R.
Right arrow Articles by Frazer, I. H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS