International Immunology Advance Access originally published online on January 23, 2006
International Immunology 2006 18(3):435-444; doi:10.1093/intimm/dxh383
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Antigen dose governs the shaping of CTL repertoires in vitro and in vivo
1 Division of Molecular Life Sciences and College of Pharmacy, Ewha Womans University, 11 Daehyundong, Seoul 120-750, Korea
2 Division of Allergy, Asan Medical Center and Asan Institute for Life Science, University of Ulsan College of Medicine, 388-1 Pungnap-2dong, Songpa-gu, Seoul 138-736, Korea
3 Present address: Research Laboratory, Anterogen Co., Ltd., Seoul 156-811, Korea
Correspondence to: K.-Y. Lee; E-mail: kiyounglee{at}amc.seoul.kr and K. Kim; E-mail: khyounk{at}ewha.ac.kr
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Abstract |
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Although it is well established that antigen dose plays an important role in determining the quality of T cells induced in vitro, it has not well been determined whether antigen dose also affects T cell repertoires induced in vivo. This study demonstrates that variation of antigen doses in vivo as well as in vitro induce structurally and functionally different T cell repertoires. CTLs generated in vitro with a low antigen dose showed much higher T cell responsiveness than CTLs generated with a high antigen dose, and the two CTL populations employed different TCR Vß chains. This is most likely due to repertoire selection based on TCR affinity. The secondary in vivo responses with a high or low dose of antigen following the primary response raised with the same dose resulted in a reversed dominance pattern of two particular TCR Vß phenotypes. TCR affinity of these two T cell populations appeared different, suggesting avidity selection based on antigen availability. Indeed, they required a distinct level of antigen for maximal cytolytic function, implying a different functional avidity. These results suggest that antigen-specific T cell repertoire is substantially affected by the antigen dose employed in vivo as well as in vitro.
Keywords: antigen dose, CTL, repertoire development, T cell avidity
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Introduction |
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The characteristics of a T cell repertoire responsive to a given antigen are influenced by numerous parameters such as physicochemical nature of antigen, antigen dose and the intrinsic diversity of T cell repertoire available in vivo (1–5). Considerable evidences now indicate that antigen dose plays an important role in determining the quality of antigen-specific T cells since functional avidity and/or TCR affinity of responding T cells are greatly dependent on the concentration of antigen used for in vitro expansion of both CD4+ T cells (6, 7) and CD8+ T cells (8–10). A low antigen dose induced T cells with high-avidity TCR, whereas a high antigen dose generated T cells with low-avidity TCR. It has been suggested that a given range of antigen doses used for T cell activation and proliferation resulted in a T cell population with a unique TCR affinity range in consequence of the selective expansion of T cells with specific TCR affinity for a given antigen concentration (11). However, the relationship between an in vivo repertoire and an in vitro-expanded populations under varying antigen concentrations has not been well defined at TCR level.
Affinity maturation, which is a well-established phenomenon of B cell responses, is also believed to occur in T cell responses. With repeated exposures to the same antigen, it has been suggested that selective expansion of high-affinity T cells resulting from T cell competition for antigen occurs during secondary immune responses (12, 13). If the frequency of T cells responding to the same antigen exceeds the availability of antigen-presenting cells (APCs), T cells with high-affinity TCR would restrict the response of T cells with low-affinity TCR by prior occupation of TCR-binding site on the APCs (14–16). In this regard, antigen dose used for in vivo immunization may also have a role in regulating T cell responses. Since limited availability of an antigen is likely to provoke competition between T cells for the antigen, alteration of antigen dose might contribute to the shaping of T cell repertoire by changing antigen density on an individual APC and/or the number of antigen-bearing APCs available for T cell interaction.
Here, we examined how the variation in antigen dose influences the functional T cell avidity of in vitro-expanded populations, how well the T cell populations expanded in vitro at a certain antigen dose reflect the T cell repertoire previously established in vivo and whether the variation in antigen dose also affects in vivo selection of the T cell repertoire. We report here that differential antigen doses caused a selective expansion of CTLs in vivo as well as in vitro that could be distinguished by TCR Vß usage and functional avidity.
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Methods |
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Cells, viruses and mice
EL-4 cells were purchased from the American Type Culture Collection (Rockville, MD, USA). The cells were cultured at 37°C with 5% CO2 in RPMI 1640 medium (GIBCO BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; GIBCO BRL), 2 mM L-glutamine, 100 U ml–1 of penicillin G, 100 µg ml–1 of streptomycin, 250 ng ml–1 of amphotericin B, 10 mM HEPES, 24 mM Na2HCO3 and 50 µM 2-mercaptoethanol. Adenovirus type 5 (Ad5), kindly provided by D. Lim (KRIBB, Taejeon, Korea), was propagated in 293 cells and its plaque-forming unit (pfu) was determined as previously described (17). The viruses were stored at –70°C until use. C57BL/6 mice were obtained from Daehan Laboratory Animal Research Center (Eumsung, Korea) and maintained under specific pathogen-free conditions. Male or female mice were used for the experiments at 6–8 weeks of age.
Synthetic peptide and tetramer
A peptide (E1Bp; VNIRNCCYI) derived from the Ad5E1B region was synthesized by solid-phase synthesis. The synthetic peptide was purified to >95% purity by reverse-phase HPLC and dissolved in 20% dimethyl sulfoxide at a concentration of 10 mM. MHC tetramer of H-2Db/E1Bp was synthesized and provided by MHC Tetramer Core Facility, National Institute of Allergy and Infectious Disease (Bethesda, MD, USA). Before using, the tetramer was tested for its antigen-specific binding and titrated against appropriate CTL clones to determine the dose that induce maximal staining.
Viral infection and peptide immunization
Groups of mice (two or three mice per group) were intra-peritoneally injected with 1 x 1010 pfu of Ad5. For peptide immunization, mice were injected at footpad with 300 or 5 µg peptide in incomplete Freund's adjuvant (IFA) 30 days after primary Ad5 immunization.
Generation of CTL lines and CTL clones
Spleens were removed from mice 2–3 weeks after Ad5 immunization and generated into single-cell suspension in HBSS. After washing with HBSS, RBCs were lysed by incubating with RBC lysis buffer (0.017 M Tris base and 0.144 M NH4Cl) at 37°C for 5 min. The resulting splenocytes were cultured with the indicated concentrations of E1Bp to induce polyclonal E1Bp-specific CTLs. In order to generate CTL lines, re-stimulation with syngeneic splenocytes treated with 50 µg ml–1 of mitomycin-C (Sigma) in the presence of the peptide and 2.5% concanavalin A-activated rat spleen culture supernatant (CAS), as a source of IL-2, was done weekly. CTL clones were established by limiting dilution of the CTLs stimulated for 2–3 weeks. Briefly, the resulting CTLs were plated in 96-well round-bottom plates containing mitomycin-C-treated syngeneic splenocytes (2 x 105 cells), peptide and 2.5% CAS at 0.3–5 cells per well. When the frequency of growing cells at a given cell dilution was <20%, the proliferating cells on the culture plate were chosen for further expansion and tested for antigenic specificity using a standard cytotoxicity assay. The CTL clones obtained were maintained by weekly re-stimulation.
Cytotoxicity assay
Cytotoxicity was measured by a standard 4-h 51Cr-release assay. Target cells were labeled with 25 µCi ml–1 of 
(NEN, Boston, MA, USA) for 1 h at 37°C, washed with HBSS and pulsed with the peptide at the indicated concentrations for 1 h in RPMI 1640 medium. The washed target cells were plated with effector cells on a microtiter assay plate at an indicated effector : target (E : T) ratio and incubated at 37°C for 4 h. Radioactivity in the supernatant was then determined by 
-radiation counting. The specific lysis was determined by using the formula [(experimental release – spontaneous release)/(maximum release – spontaneous release) x 100]. All experiments were performed in triplicate and the spontaneous release was always <10% of the maximum release. To block CD8 molecules, indicated amounts of purified anti-CD8 antibody (PharMingen, San Diego, CA, USA) were added to effector cells 30 min prior to the incubation with target cells. Purified anti-CD4 antibody (PharMingen) was used as a negative control. The inhibition was determined using the formula [1 – (percent lysis in the presence of antibody/percent lysis in the absence of antibody) x 100].
Intracellular IFN- assay
An intracellular IFN- assay was performed as described previously (18). The CTLs (5 x 105 cells), harvested after repeated antigenic stimulation, were incubated with EL-4 cells (1 x 106 cells) pulsed with various concentrations of the peptide in the presence of a protein transport inhibitor, monensin (PharMingen) for 5 h at 37°C. After incubation, cells were washed with PBS containing 0.5 mM EDTA and stained with FITC-conjugated anti-CD8 antibody (PharMingen) for 30 min on ice. After washing with PBS, the cells were treated with PBS containing 4% PFA for 5 min at 37°C and washed once with PBS containing 1% BSA. After two additional washes with PBS (0.1% BSA, 0.1% saponin and 0.01 M HEPES), the cells were stained with PE-conjugated anti-IFN- antibody (PharMingen) for 30 min at room temperature and washed twice with PBS (0.1% BSA, 0.1% saponin and 0.01 M HEPES). Cells were analyzed with a flow cytometer (FACSCalibur apparatus; BD Bioscience, San Jose, CA, USA).
Vß usage analysis
To examine TCR Vß usage of the CD8+ T cells secreting IFN-, splenocytes (2 x 106) obtained from the Ad5-immune mice were incubated with the peptide, 20 U ml–1 of human IL-2 and monensin for 5 h. After incubation, the cells were treated in the same manner as described above except that the first staining step was performed with a panel of FITC-conjugated anti-TCR Vß antibodies (PharMingen) and Cy-Chrome-conjugated anti-CD8 antibody (PharMingen). For analysis of TCR Vß chain usage by using tetramer, splenocytes freshly isolated from immune mice or CTL lines were washed with FACS buffer (PBS containing 2% FBS and 0.1% sodium azide) and then stained with FITC–anti-TCR Vß antibodies, PE–tetramer, and Cy-Chrome–anti-CD8 antibody for 30 min at ice. After gating on forward scatter/side scatter channel (FSC/SSC) parameter, percentage of T cells bearing each TCR Vß chain and their tetramer staining intensity were analyzed by flow cytometry.
TCR Vß5.1/2+ and Vß8.1/2+ T cell purification
Splenocytes were obtained from the Ad5-immune mice and cultured with 200 pM of the peptide to generate E1Bp-specific CTLs. TCR Vß5.1/2 or Vß8.1/2 cells were purified from the E1Bp-specific CTL population by using MiniMACS magnetic separation system (Miltenyi Biotec, Sunnyvale, CA, USA) according to the manufacturer's instructions.
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Results |
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Inverse relationship between antigen dose and in vitro T cell responsiveness
Antigen dose has been reported to affect functional avidity of the T cell populations induced in vitro (6–10). First, we here attempted to examine the relationship between antigen dose and T cell responsiveness under in vitro conditions, employing adenovirus-specific CTL lines. In our previous study we showed that E1Bp, an Ad5E1B-encoded epitope, served as one of the immunodominant epitopes in C57BL/6 mice immunized with Ad5 (19). Splenocytes from C57BL/6 mice immunized with 1 x 1010 pfu of Ad5 were stimulated with a high (2 µM), intermediate (0.02 µM) or low (0.0002 µM) dose of the E1Bp peptide. Figure 1 shows the cytolytic activities of the splenocytes cultured in the presence of E1Bp against EL-4 target cells pulsed with various concentrations of the peptide. CTLs generated with a high antigen dose lysed targets sensitized with antigenic peptide at concentrations >1 nM. In contrast, CTLs generated with a low antigen dose required a pulsing antigen concentration of <10 pM to lyse the targets. Thus, the high dose-induced CTLs required >103-fold higher peptide concentration than the low dose-induced CTLs to sensitize targets. These results were not likely due to a difference in the number of antigen-specific CTLs induced during the in vitro stimulation, since after three rounds of in vitro stimulation, 80–90% of the CD8+ T cells were E1Bp-specific and the number of responding cells in each populations was similar, as determined by the intracellular IFN-
assay (data not shown). Similar results were obtained from three independent groups of T cell populations. In accordance with the previous observations (8–10), these results indicate that the antigen dose used for in vitro expansion of CTLs was closely related to the quality of the resulting population; antigen dose was inversely related to T cell responsiveness.
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Functional avidity of CTLs induced in vitro is a function of their TCR affinity
CD8 molecule, which is known to enhance the intrinsic TCR affinity by stabilizing the interaction between TCR and MHC–antigen complex, may not always be crucial to mounting T cell responses. While CTLs with low-affinity TCR require CD8 involvement to elicit T cell response, high-affinity TCRs recognize their ligands in a CD8-independent manner. Therefore, CD8 dependency in T cell antigen recognition has been used to estimate TCR affinity indirectly (20–22). The sensitivities to CD8 blocking of the two T cell populations cultured with either a high or a low dose of peptide were examined. Treatment with anti-CD8 antibody markedly inhibited the cytolytic activity of the high dose-induced CTLs in a dose-dependent manner, whereas cytolytic activity was not significantly inhibited in the low dose-induced CTLs even in the presence of a sufficient amount of anti-CD8 (Fig. 2A). These results suggest that TCR affinity of the low dose-induced CTLs was much higher than that of the high dose-induced CTLs.
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Since it has previously been shown that CD8 dependency of CTL function was greatly influenced by antigen density expressed on APCs (23, 24), CTL sensitivity to CD8 blocking was also analyzed with varying antigen concentrations in the presence of a saturating amount of anti-CD8 antibody. The cytolytic activity of the high dose-induced CTLs was substantially reduced even with a high concentration (10 µM) of the pulsing peptide as shown in Fig. 2(A) and almost completely inhibited at 10 nM of the peptide, while the low dose-induced CTLs were not affected at high peptide concentrations and only marginally affected at an extremely low concentration (10 pM) of the peptide, implying greatly different TCR affinity between the two CTL populations (Fig. 2B). Collectively, these results suggest that the different cytolytic capabilities of the in vitro-expanded CTL populations in the presence of varying doses of antigenic peptide are most likely due to the generation of T cells bearing different TCR affinities for the peptide–MHC complexes since no differences in TCR or CD8 expression levels were observed (data not shown).
Distinct functional avidity between the high dose- and low dose-induced CTL clones
To ascertain the influence of antigen dose on the functional avidity of the in vitro-expanded CTL populations, a panel of antigen-specific CTL clones was established from the high dose- or low dose-induced CTL populations. Cytotoxicity assays were performed with the CTL clones at various antigen concentrations. Although the maximum responses were comparable, differences in the overall sensitivity of antigen recognition between the high dose- and low dose-induced CTL clones were apparent (Fig. 3A). Consistent with the results mentioned above (Fig. 2), the low dose-induced clones required an 
103-fold lower peptide concentration than the high dose-induced clones to lyse target cells to a similar extent. Likewise, the high dose-induced clones appeared more sensitive to blocking by anti-CD8 antibody (Fig. 3B). Cytolytic activities of the high dose-induced clones were almost completely blocked by anti-CD8 antibody, although interclonal variations in the sensitivity were observed, while cytolytic activities of the low dose-induced clones were hardly inhibited by the same amounts of antibody. Furthermore, these results were confirmed by testing the capacity of the clones to secrete IFN- in response to varying concentrations of the peptide by flow cytometry, which allows evaluation of the responsiveness of individual CD8+ T cells (data not shown). In this assay, the high dose- and low dose-induced clones required markedly different levels of antigen density to exert equivalent responses, whereas the percentage of cells secreting IFN- in the presence of a sufficient amount of antigen was similar between the two groups. These results suggest that different capability in antigen recognition was due to the difference in the antigenic sensitivity of the T cells, not the number of responding T cells.
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E1Bp-specific T cell repertoire induced in vivo was developed into unexpected sub-populations during in vitro culture
As shown above, individual T cells generated with varying antigen doses showed markedly different functional avidity and TCR affinity. We previously reported that E1Bp-specific T cell repertoire was quite diverse at the level of both functional specificity and its TCR Vß usage (19). Therefore, it seems likely that polyclonal T cell populations induced by Ad5 immunization could be further selected into distinctive sub-populations based on TCR affinity during in vitro stimulation, especially under a particular antigen dose. To evaluate this possibility, TCR Vß usage of the E1Bp-specific T cells generated by in vivo immunization was compared with that of the T cell populations obtained by in vitro stimulation following immunization. Splenocytes were prepared from C57BL/6 mice immunized with 1 x 1010 pfu of Ad5, and an aliquot was subjected to analysis for TCR Vß usage. The E1Bp-specific CTLs were triply stained with anti-CD8 antibody, anti-IFN-
antibody and a panel of anti-TCR Vß antibodies before they were analyzed by flow cytometry. The remaining splenocytes were divided into two parts and subjected to in vitro expansion with either a high (2 µM)- or low (0.0002 µM)-dose antigen. After three or four rounds of in vitro culture, TCR Vß profiles of the high dose- or low dose-induced CTLs were determined by using anti-TCR Vß antibodies. These experiments enabled us to evaluate how the E1Bp-specific T cell repertoire induced in vivo by virus challenge would be modified during in vitro culture under a particular antigen concentration. The E1Bp-specific T cell populations induced in vivo-expressed TCR Vß8.1/2 predominantly and TCR Vß5.1/2 to a lesser extent (Fig. 4). Interestingly, the two resulting CTL populations obtained from subsequent in vitro cultures with different antigen concentrations showed very distinct TCR Vß profiles. The CTLs cultured in vitro in a low antigen concentration were stained predominantly by anti-TCR Vß5.1/2 antibody, and to a lesser extent, by anti-TCR Vß8.1/2. Although the predominance of the two Vß segments was reversed as compared with the TCR Vß profile of the ex vivo repertoire, the two Vß families remained dominant. In contrast, the Vß repertoire of the CTLs cultured in a high antigen concentration was markedly changed; most T cells expressed TCR Vß12 chain, which constituted only a minimal fraction of the E1Bp-specific T cell repertoire in vivo. In Db/E1Bp-tetramer-binding assay, these TCR Vß12+ cells were shown to specifically bind with Db/E1Bp-tetramer (data not shown). These observations, in the context with the results of Figs 1 and 2, suggest that Vß5.1/2+ and Vß8.1/2+ populations expanded at a low antigen concentration is responsible for the high functional avidity, and Vß12+ T cells expanded at a high antigen concentration is responsible for the low functional avidity. It has previously been demonstrated that high-avidity CTLs undergo deletion with high antigen dose, leaving only low-avidity CTLs (11, 24). Therefore, the predominance of Vß12 phenotype in the presence of a high antigen dose might be due to both deletion of the high-avidity T cells expressing Vß5.1/2 or Vß8.1/2 and rapid proliferation of the low-avidity T cells expressing Vß12. These results collectively show the impact of antigen dose on in vitro selection of E1Bp-specific T cell repertoire thereby promoting the induction of CTL responses with different efficiency.
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The role of antigen dose in shaping the in vivo T cell repertoire
The finding that antigen dose used for in vitro T cell expansion plays a crucial role in the selection of functionally and structurally distinct T cell subsets prompted us to investigate whether this relationship is applicable to in vivo conditions. To investigate the influence of antigen dose used for in vivo priming on the characteristics of the E1Bp-specific T cell populations generated, Ad5-primed mice were re-challenged with different doses of E1Bp peptide. However, it has previously been reported that immunization with the E1Bp peptide in C57BL/6 mice led to induction of tolerance to E1Bp-specific CTLs associated with functional deletion of peptide-specific CTLs (25). We therefore first examined whether E1Bp-specific CTL responses could be properly induced by the subsequent E1Bp immunization of the Ad5-primed mice. C57BL/6 mice that had been primed with Ad5 (1 x 1010 pfu) were re-challenged with a high dose (300 µg) or a low dose (5 µg) of E1Bp in IFA, and the resulting immune splenocytes were analyzed for their ability to produce intracellular IFN-
in response to E1Bp. As shown in Fig. 5, E1Bp-specific CTLs turned out to be induced in the Ad5-primed mice by boosting with E1Bp peptide when compared with the PBS-injected mice. Even when administered with the peptide as much as 300 µg, tolerance to the E1Bp-specific CTLs was not observed, indicating that in contrast to naive mice, Ad5-primed mice could efficiently elicit E1Bp-specific CTL response rather than tolerance induction by peptide administration.
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Next, in vivo T cell repertoires induced with a different amount of peptide were examined. Since in vitro manipulation of T cells could result in some distortion of T cell responses previously established in vivo as observed above, we directly analyzed the antigen-specific T cell repertoire generated in vivo without in vitro stimulation by using a H-2Db/E1Bp-tetramer. Ad5-primed mice were re-challenged with a high or a low dose of peptide as described in Fig. 5, and splenocytes obtained from these mice were triply stained with mAb to CD8, TCR Vß and the tetramer.
The E1Bp-specific CTL populations of the peptide-rechallenged mice appeared to express predominantly Vß5.1/2 and Vß8.1/2 phenotype, similar to those observed in the primary Ad5-immune mice, and the percentage of the T cells bearing these two TCR Vß chains were represented in Fig. 6(A). The predominance between Vß5.1/2 and Vß8.1/2 in the two groups of mice (high dose and low dose) observed was significantly different. E1Bp-specific T cells from the mice re-challenged with high-dose peptide expressed Vß5.1/2 predominantly. In contrast, Vß8.1/2 phenotype was predominant in the T cells obtained from the low-dosed mice. In non-infected mice as a control, percentages of either CD8+Vß5.1/2 Db/E1Bp-tetramer+or CD8+Vß8.1/2 Db/E1Bp-tetramer+were below 2% (data not shown). The results shown above suggest that there are some differences in the quality of Vß5.1/2+ and Vß8.1/2+ T cells since in vitro-expanded T cells showed different functions depending on antigen dose used.
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To examine this possibility, Vß5.1/2+ and Vß8.1/2+ T cells were analyzed for the efficiency to recognize their ligand and the capacity to induce effector function. Firstly, tetramer-binding intensity of both CD8+Vß5.1/2+ and CD8+Vß8.1/2+ cells obtained from the peptide-rechallenged mice was measured. It has well been documented that tetramer-binding efficiency of an individual T cell correlates with the TCR affinity (14, 26). In addition, TCR affinity of a given T cell is a crucial parameter for determining functional avidity of T cell for its ligand. As shown in Fig. 6(B), mean fluorescence intensity of Vß5.1/2+ T cells stained with the tetramer was 80.7 and that of Vß8.1/2 T cells was 120.6, suggesting that the overall TCR affinities of T cells expressing Vß8.1/2 phenotype was relatively higher than those of Vß5.1/2. The result is able to arise from the possibility that the Vß-specific mAbs may partially block the tetramer staining. To confirm this possibility, the cells were sequentially stained first with anti-CD8 antibody, and anti-Vß-specific antibodies either anti-Vß5.1/2 or anti-Vß8.1/2, then with the tetramer. The result was consistent with Fig. 6(B), suggesting that Vß-specific mAbs both Vß5.1/2 and anti-Vß8.1/2 does not affect on the tetramer staining (data not shown). Furthermore, when we checked the levels of CD3 molecules to confirm the possibility that TCR complexes may be internalized during the staining procedures, any difference in each other could not be observed (data not shown). Similar results were observed in both 300- and 5-µg rechallenged mice. In addition, this difference was more evident in E1Bp-specific CTL population obtained from in vitro culture (Fig. 6C). T cells bearing TCR Vß8.1/2 showed significantly high affinity compared with that of Vß5.1/2+ T cells. Secondly, to further examine the functional difference between the two TCR phenotypes, each of them was separated from the E1Bp-specific CTL population used in the experiment of Fig. 6(C) by using magnetic separation columns. Each of the resulting subsets included <1% of the other cell type (data not shown). When cytotoxicity assay was performed, Vß8.1/2+ T cells lysed antigen-pulsed targets more efficiently than Vß5.1/2+ T cells (Fig. 7). Taken together, these data demonstrate that in vivo immunization with varying antigen dose can influence composition and function of T cell repertoire by preferential expansion of a particular T cell subset with a specific TCR affinity range.
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Discussion |
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In this study, the influence of antigen dose on immune responses was studied with the E1Bp-specific CTL repertoire induced with Ad5 both in vivo and in vitro. In accordance with the previous observations, T cell populations cultured in vitro with varying concentrations of the E1Bp peptide greatly differed in quality: a high antigen dose generated CTLs with low avidity, whereas low antigen dose-induced CTLs displayed high functional avidity. This finding was also confirmed with the clones established with high or low antigen dose. Such a distinctive T cell responsiveness is most likely due to changes in the repertoire of the high dose- and the low dose-induced T cell populations, presumably in terms of TCR affinity. Interestingly, the TCR Vß repertoire of T cells induced in vitro with high antigen dose was completely different from the in vivo-induced T cell repertoire, whereas the in vitro-expanded T cells with low antigen dose appeared to be similar to that of in vivo-induced T cells. These observations suggest that functional avidity of E1Bp-specific T cell repertoire covers a quite broad range and functional avidity of selected T cells seems to be inversely proportional to antigen dose used during in vitro culture. Furthermore, it also suggests that though it contributes little to overall immune response at the time of in vivo priming with a given antigen dose, a particular sub-population is able to selectively proliferate in vitro under some antigen dose conditions.
In vivo expansion of a T cell subset also appeared to be influenced by antigen dose used for re-challenge. E1Bp-specific T cell repertoire induced by virus immunization consisted of TCR Vß8.1/2 T cells as a dominant population and Vß5.1/2 T cells as a sub-dominant population at the peak of the primary response, and this composition of the repertoire was maintained in the memory pool. After re-challenge with the antigenic peptide, the two T cell subsets bearing either TCR Vß5.1/2 or 8.1/2 were differentially expanded with different antigen doses. Furthermore, it was revealed that they differed in mean TCR affinity and functional avidity as observed with the T cells expanded in vitro with varying antigen concentration. It is therefore very likely that antigen dose played an important role in shaping of in vivo T cell repertoire, as in vitro repertoire was affected by varying antigen concentrations.
However, no outgrowth of TCR Vß12+ T cell subset with low TCR affinity, which occurred during in vitro expansion with high antigen dose, was observed in the peptide-challenged mice. This might be attributed to the difference in the actual amount of antigen available in vivo and in vitro. A high antigen dose given in vivo might not have accomplished an antigen density on the APCs high enough to recruit Vß12 T cells of low affinity and not Vß5.1/2 and 8.1/2 T cells of high affinity. Alternatively, it is possible that the role of antigen dose in vivo is less likely significant than that of in vitro because of the substantial differences in the immunological environment between in vivo and in vitro such as the number of available APCs, accessibility to antigen, physiological structure of T cell–APC interaction and another cellular and soluble components regulating immune response.
Previous studies suggested that a specific range of antigen dose was required for optimal proliferation of T cell subset with a particular avidity (1, 11). Data from in vivo study as well as in vitro also support this notion. In this regard, it is interesting to note that the degree of T cell diversity to a given antigen may be much greater than that has been observed experimentally. Highly diverse repertoires, as well as repertoires limited to specific TCR 
- and ß-chains have been described (26–32). However, the data might not have accurately reflected the potential diversity of the T cell repertoire since only a single antigen dose for in vivo priming and/or in vitro expansion had been employed in the previous experimental systems. Re-evaluation of previous results demonstrating TCR repertoires specific for single antigenic peptides may be necessary.
Taken together, this study demonstrates that different antigen doses caused selective expansion of CTLs with different functional avidity in vivo as well as in vitro. Hence, a given antigen-specific T cell repertoire can be characterized in terms of functional avidity as well as antigenic specificity. As diversity in antigenic specificity is necessary to cope with the immune escape of a pathogen by mutation, maintenance of a repertoire with diverse avidity might also be required to mount effective immune responses against diverse antigen loads. Insight into the relationship between antigen dose and repertoire selection will advance the understanding of CD8+ T cell-mediated immune responses.
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Acknowledgements |
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This work was supported by Korean Research Foundation (KRF-2003-015-C00509) and by a grant (2006-397) from the Asan Institute for Life Science, Seoul, Korea.
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Abbreviations |
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| Ad5 | adenovirus type 5 |
| APC | antigen-presenting cell |
| CAS | concanavalin A-activated rat spleen culture supernatant |
| E : T | effector : target |
| FBS | fetal bovine serum |
| IFA | incomplete Freund's adjuvant |
| pfu | plaque-forming unit |
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Notes |
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Transmitting editor: E. Vivier
Received 28 July 2005, accepted 12 December 2005.
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