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International Immunology Advance Access originally published online on March 21, 2007
International Immunology 2007 19(4):497-507; doi:10.1093/intimm/dxm016
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Avidity of CD8 T cells sharpens immunodominance

Amiran H. Dzutsev1, Igor M. Belyakov1, Dmitry V. Isakov1, David H. Margulies2 and Jay A. Berzofsky1

1 Vaccine Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-1578, USA
2 Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

Correspondence to: I. M. Belyakov; E-mail: igorbelyakov{at}yahoo.com; and to J. A. Berzofsky; Email: berzofsk{at}helix.nih.gov


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
In the course of viral infection, the immune system exploits only a fraction of the available CTL repertoire and focuses on a few of a myriad of potentially antigenic peptides. This phenomenon, known as immunodominance, depends on a number of factors, including antigen processing and transport, MHC binding, competition for antigen-presenting cells, availability of the CD8 T cell repertoire and other mechanisms that function largely by restricting the immune response. Here we elucidate a novel mechanism that increases the immunodominance of the epitope rather by enhancing the immune response. Using a peptide-specific MHC-restricted mAb and functional assays of CTL activation, we show that T cells with high avidity for the immunodominant, H-2Dd restricted, P18-I10 epitope expand rapidly following immunization, and this expansion in turn determines the level of the P18-I10 epitope immunodominance. This proliferation has little dependence on the number of MHC–peptide complexes. Since most self-reactive T cells of high avidity are depleted in the thymus, the selection of immunodominant epitopes based on the expansion of high-avidity T cells in the periphery reduces the potential for autoimmunity.

Keywords: Immunodominance, cytotoxic, T lymphocytes, avidity, epitope


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Supplementary data
 References
 
Amidst a sea of different foreign and self-epitopes, the immune system targets just a few of them (1–4). This phenomenon is known as immunodominance. Despite considerable study of CD8 T cells, the mechanistic basis of immunodominance of CTL is still incompletely understood. All the main elements of the adaptive immune system, B cells (5), CD4+ (6) and CD8+ T cells have been shown to be very discriminating in the antigens that they recognize, and CD8+ T cells seem to be particularly so (7–10). Many factors were shown to be involved in development of immunodominance. We may consider the generation of the immunodominant response in two stages. The first would include all those processes that involve presentation of the epitopes to the T cells, such as many steps of antigen expression and processing (11), transport of antigenic peptides to the endoplasmic reticulum (ER) (12, 13), the efficiency of delivery to and loading of MHC-I molecules, the efficacy of MHC expression at the cell surface and the affinity and stability of the peptides for the available MHC molecules (8, 14–16). A second stage would include those mechanisms and factors that underlie subsequent events, such as available CD8 T cell repertoire (2, 17–19), the competition of CD8 T cells for antigen-bearing cells and the timing of T cell recruitment (20). The outcome of the events that happens during the first stage affects not only the breadth of the numbers of different epitopes presented but also the level of their expression in a complex with MHC molecules on the surface of antigen-presenting cells (APCs). A number of studies have investigated the degree of dependence of immunodominance of the epitope on the number of MHC–peptide complexes presented to the T cells. Interestingly, although, in general, immunodominant peptides as a group usually have a high MHC-binding affinity (21–25), there seemed to be no tight correlation between binding affinity and immunodominance (26). Nevertheless, one would expect that those peptides that tend to elicit low-avidity T cells might be able to recruit more cells if more MHC–peptide complexes are generated and therefore increase the immunodominance of the epitope. Attempts to over-express MHC–peptide complexes to compensate for sub-dominant epitopes have not been fruitful (27). Several studies have tried to measure directly the relation between the number of MHC–peptide complexes on the APC and the CD8 T cell immune response using specific MHC–peptide antibodies (25, 27–30). These studies, in several laboratories, based on the H-2Kb-SIINFEKL MHC–peptide complex, suggested that increased expression leads to an increase in the overall immune response, but not an increase in immunodominance. However, the moderate level of immunodominance of the ovalbumin epitope (~5% of the total anti-viral response, if expressed in vaccinia virus) could significantly narrow the window for the research of possible fluctuations in the immunodominance of the epitope. In our research model, we used a much more dominant H-2Dd-restricted gp120 HIV-derived epitope, P18-I10, which represented ~25% of total anti-viral response. In combination with a novel sensitive method of estimating the number of P18-I10–H-2Dd complexes on the surface of the virally infected cells, it presented an opportunity to study these questions with a higher degree of sensitivity.

Here we demonstrated that a significant increase in the level of the MHC–peptide complexes of the dominant epitope on the surface of APCs does not increase further the level of immunodominance to it. We also showed that the immunodominance is related to the ability of the high-avidity CD8+ T cell sub-population to expand much more rapidly than the low-avidity one. We define functional avidity for this purpose by the ability of the T cells to respond in an antigen titration to lower (high avidity) or higher (low avidity) concentrations of antigen. The functional avidity is distinct from the thermodynamic affinity of the TCR for the peptide–MHC complex, which is one important component of avidity; however, functional avidity as used here is also dependent on TCR density, CD8 density, signaling efficiency and probably other factors. Our results suggested that high-avidity T cells contribute much more to the development of the immune response than low-avidity T cells. Our results also indicate that the level of the immunodominance to the epitope is directly proportional to the percent of high-avidity T cells specific for the epitope.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Supplementary data
 References
 
Mice, cell isolation and cell lines
BALB/cJ, B10.D2/nsn, DBA/2J, LG/J and B6.C-H2d, 4–6-weeks old, were purchased from Jackson Laboratories. BALB/c mice for some sets of experiments were obtained from Frederick Cancer Research Facility (Frederick, MD, USA). At different time points after immunization, cells were isolated from the spleen and erythrocytes were lysed by ACK-lysing buffer, Cambrex. All manipulations were performed in cold 2% FCS RPMI1640 media. P815 cells were grown in complete 10% FCS media. Only adherent P815 cells were used for all assays. Dendritic cells (DCs) were grown from bone marrow by cultivating in 1µg ml–1 of granulocyte macrophage colony-stimulating factor in complete 10% FCS media for 5 days. Before any manipulations, dead cells were removed with a Dead Cell depletion kit from Miltenyi Biotec.

Viruses and peptides
VSC8 virus (31) is a thymidine kinase-negative attenuated vaccinia virus. vPE16 virus (32) expresses gp160 IIIB determinant of HIV that contains the H-2Dd-restricted CTL epitope RGPGRAFVTI (P18-I10) (33, 34). These were kindly provided by P. Earl and B. Moss. The minigene P18 vaccinia virus (35), contains only the P18 coding sequence linked with the adenovirus E19 leader sequence targeting the synthesized peptide to the ER, from J. Gibbs, J. Yewdell and J. Bennink. Both constructs are under the early-late vaccinia promotor. DCs and P815 cells with >99% of viability were infected with the viruses in concentration 107 cells ml–1 in 0.5% BSA at a multiplicity of infection of 50 placque-forming units per cell for 30 min. Afterwards, a 10x volume of 10% FCS-supplemented media was added to the samples and incubated for additional 18–24 h. Both vaccinia constructs yielded infection of a similar number, ~0.5%, of ovary cells at day 5 after infection (AH Dzutsev, IM Belyakov, D Isakov, S Gagnon, DH Margulies, and JA Berzofsky, in preparation). P18-I10 peptide gp160 HIV IIIB was purchased from Multiple Peptide Systems (San Diego, CA, USA) at >95% purity.

Immunizations
Mice were immunized once intra-peritoneally with 107 pfu of sonicated viruses in 100 µl of sterile PBS.

ELISPOT assay
Nitrocellulose plates (Millipore) were incubated overnight with capture anti-IFN-{gamma} antibody (MABTECH). Then plates were washed and blocked for 1 h with 10% FCS media. The ELISPOT assay with virus-infected cells was described previously (36). Target P815 cells had been previously pulsed with peptides or infected with vaccinia virus for 5 h, washed twice and UV irradiated for 15 min. Then target and effector cells were plated in round-bottomed 96-well plates, centrifuged at 500 x g for 1 min, incubated for 30 min at 37°C and then transferred to the ELISPOT plate. After 24 h of co-cultivation, spots were developed by biotinylated anti-IFN-{gamma} antibody (MABTECH), Vectastain ABC kit (Vector Laboratories) and an AEC, 3-amino-9-ethylcarbazole, substrate kit (Vector Laboratories), according to the manufacturer’s instructions. For estimation of the percent of high-avidity T cells, P815 cells were pulsed with 1 nM or 1 µM of P18-I10 peptide. The level of high-avidity T cells was calculated as the percent of cells responding to 1 nM peptide compared with cells responding to 1 µM of P18-I10 peptide.

Antibodies, tetramer assays and flow cytometry
All flow cytometry samples were analyzed on a FACSCalibur (Becton Dickinson). P18-I10 complexes on the surface of infected cells were analyzed with KP15 mAb, which specifically recognizes the H-2Dd–P18-I10 peptide complex (37), by a three-step amplification method with biotinylated secondary antibody (BD PharMingen) and Cy5-labeled streptavidin (BD PharMingen). To quantify surface levels of H-2Dd–P18-I10, DCs were infected with minigene P18 vaccinia and stained with saturating amounts of primary KP15 mAb, so that one IgG molecule should bind only one MHC–peptide complex. Then cells were stained with saturating amounts of secondary FITC-conjugated anti-mouse IgG. In parallel, the same PE-labeled secondary IgG antibody was used to stain calibration beads (DAKO) coated with a known number of mouse IgG molecules which were later used to construct a standard curve of the number of IgG molecules versus mean fluorescence intensity (MFI). The calibration curve was used to estimate number of H-2Dd complexes per cell by comparing it with the MFI of the samples. To calculate numbers of MHC-P18–I10 complexes on the cells infected with vPE16 virus, we stained minigene P18 vaccinia- and vPE16-infected cells with three-step amplification method and used obtained ratios of MFI and known numbers of MHC–peptide complexes obtained earlier for minigene P18 vaccinia-infected cells to estimate numbers of MHC–P18-I10 complexes for vPE16-infected cells. We also noted that increasing the input of virus pfu did not increase the density of peptide–MHC complexes, but did increase the number of cells infected, suggesting that the number of virus particles taken up per cell did not change.

For intracellular protein detection, cells were permeabilized with Perm/Fix and Wash buffers (BD PharMingen) and stained with primary anti-gp160 mAb, a gift of Bernard Moss, National Institute of Allergy and Infectious Diseases (NIAID), with secondary FITC-labeled anti-mouse IgG mAb and FITC-labeled anti-ß-galactosidase, Abcam, for virus-infected cells; and with PE-labeled anti-IFN-{gamma} antibody for immune splenocytes. P18-I10-H-2Dd tetramer was provided by the NIAID Tetramer Core Facility. For analysis of Vß chain composition, we used a Vß screening kit (BD PharMingen). For tetramer decay analysis, spleen cells were stained with P18-I10 tetramer, which was filtered through a 0.22-µm filter (Millipore) prior to the staining, washed twice with 0.5% BSA PBS and incubated at 4°C with 25 µg ml–1 of 34-5-8S mAb anti-H-2Dd to prevent rebinding. Every 10 min, samples were analyzed by flow cytometry. The filtration of the tetramer and use of a Dead Cell depletion kit (Miltenyi Biotec) prior to the staining greatly reduced background tetramer staining. For detection of high- and low-avidity T cells using tetramer, splenocytes were incubated with antibodies and tetramer–PE for 30 min, washed and incubated with excess of 34-5-8S mAb anti-H-2Dd for 1–9 h at 10°C, and then cells were washed again and stained with tetramer labeled with APC. If necessary, cells were further stained for intracellular markers. For 5-bromo-2-deoxyuridine (BrdU) staining, mice were immunized with minigene P18 vaccinia virus and were given daily newly prepared filtered BrdU (Sigma–Aldrich), 0.8 mg ml–1 in drinking water. BrdU was kept in the dark. Spleen cells were isolated at day 5. Cells were stained for surface markers and, as described elsewhere, stained for BrdU content with FITC-labeled antibodies from a BrdU kit (BD PharMingen). For the carboxyfluorescein succinimidyl ester (CFSE) proliferation assay, immune splenocytes were stained with 1 µM CFSE for 3 min in 0.5% BSA PBS and cultivated in 2% FCS RPMI1640 media in the presence of anti-CD3 mAb for 72 h, and then stained with surface markers and analyzed by flow cytometry.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Supplementary data
 References
 
An immune response to P18-I10 can be elicited by immunization with either a recombinant vaccinia virus, vPE16, which encodes the full-length envelope, or with a minigene P18 vaccinia virus which contains only the P18 coding sequence. P18-I10 expressed in minigene vaccinia was also linked with the adenovirus E19 leader sequence targeting the synthesized peptide to the ER to further enhance presentation of the epitope.

To estimate the number of complexes per virus-infected cell, we used the KP15 mAb specific for the P18-I10–H-2Dd complex (37, 38). Since this is the first report of using KP15 to evaluate P18-110-H-2Dd complexes on the surface of virus-infected cells, we initially verified the specificity of our method. To address that question, control, vSC8 virus (Fig. 1A), vPE16 (Fig. 1B) and minigene P18 vaccinia (Fig. 1C)-infected P815 cells were stained with KP15 mAb together with antibodies to gp120 (Fig. 1A–C). Lack of staining by KP15 mAbs and anti-gp120 mAbs of vSC8-infected cells, effective staining of vPE16 by both KP15 and antibody to gp120 and staining of minigene P18 vaccinia-infected cells by KP15 but not anti-gp120 indicated the specificity of the assay. The H-2Dd–P18-I10 complex expression after infection with vPE16 or minigene P18 vaccinia viruses peaked at 12–15 h after infection (data not shown). We also estimated the number of P18-I10–H-2Dd complexes per cell using KP15 and QIFIKIT (DAKO) (Fig. 1D). The number of H-2Dd–P18-I10 complexes expressed at the surface of minigene P18 vaccinia- and vPE16-infected DCs was ~4600 and 380 complexes per cell, respectively (a 12-fold difference). An increase in the multiplicity of either vaccinia virus used to infect cells did not increase the intensity of the KP15 staining, but only the percent of cells infected (data not shown).


Figure 1
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  		Fig. 1. Minigene gp160 induces higher expression of P18-I10–H-2Dd complexes than vPE16 virus. Using KP15 mAbs, we were able to stain P815 cells (A–C) or DCs (D) for the presence of P18-I10–MHC complexes on the cell surface. KP15 mAbs were used in combination with anti-gp120 (A–C) to stain virus-infected P815 cells, multiplicity of infection = 50. VSC8 virus does not express gp160 and the minigene virus encodes only the P18-I10 portion of gp160 and therefore cells infected with these did not express gp160 (A and C, respectively) in contrast to vPE16-infected cells (B). (D) The number of P18-I10–H-2Dd complexes per cell on DCs infected with different viruses. (E) Over-expression of H-2Dd–P18-I10 complexes on minigene P18 vaccinia-infected cells did not alter the recognition of other vaccinia epitopes. vSC8-immunized Spleen cells were co-cultivated in a IFN-{gamma} ELISPOT assay with P815 cells infected with vSC8 (filled diamond), vPE16 (filled circle) and minigene P18 (filled triangle). Assay shows no significant differences between groups, n = 3; error bars represent the standard deviation between samples.

 
Interestingly, the over-expression of P18-I10 peptide in minigene P18 vaccinia-infected cells did not suppress the ability of vaccinia primed CD8+ T cells to recognize other vaccinia epitopes. That was demonstrated in an IFN-{gamma} ELISPOT assay using splenic cells from mice immunized with vSC8 virus (which does not express the P18-I10 epitope) and stimulating the cells with P815 cells infected with vSC8, minigene P18 vaccinia or vPE16 viruses (Fig. 1E). We observed no difference in the number of spots regardless of the stimulator virus, an indication that vaccinia virus-infected cells in the presence of competition from the over-expressed P18-I10 epitope still retain a sufficient level of other vaccinia epitopes.

To estimate the immunodominance of the P18-I10 epitope in mice immunized with vPE16 and minigene P18 vaccinia viruses, the numbers of virus-specific CD8 T cells and the number of P18-I10-specific T cells were measured by an IFN-{gamma} ELISPOT assay (Fig. 2A and B). The numbers of P18-I10-specific CD8+ T cells induced by each virus were similar whether measured by ELISPOT or by flow cytometry after staining splenocytes with a P18-I10-H-2Dd tetramer (Supplementary Figure 1A, available at International Immunology Online). To compare the levels of immunodominance of the P18-I10 epitope in these mice, a variable, ‘ID’, representing the percent of cells specific for P18-I10 among the total virus-specific T cells, was introduced (Fig. 2C). Thus, we are examining the immunodominance of this epitope relative to all the epitopes in the whole virus.


Figure 2
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  		Fig. 2. Minigene P18 vaccinia and vPE16 viruses induced the same level of immunodominance of the P18-I10 epitope in BALB/c mice. Mice were immunized with minigene P18 vaccinia virus (A) or vPE16 virus (B) and splenocytes were analyzed at different time points, n = 3; error bars represent standard deviation between the samples. A typical experiment of four is shown. (A and B) IFN-{gamma} ELISPOT assay. Immune response to P18-I10 peptide pulsed or minigene P18 vaccinia-infected P815 cells in minigene P18 vaccinia-immunized mice (A) and vPE16-immunized mice (B). (C) Level of immunodominance of P18-I10 in vPE16 or minigene P18 vaccinia-immunized mice.

 
The level of immunodominance of the P18-I10 epitope in both groups of mice was identical throughout the entire duration of the experiment (Fig. 2C), as was estimated by measurement of the immune response (Fig. 2A and B) to P18-I10 peptide (showed in circles) and to minigene P18 vaccinia-infected P815 cells (showed in squares) and subsequent calculation of ID. The result supports previous findings (27, 28) that demonstrated lack of increase of immunodominance after over-expression of the epitope. Despite an equal level of immunodominance of the P18-I10 epitope in minigene P18 vaccinia- and vPE16-immunized mice, the functional quality of the recruited P18-I10-specific CD8 T cells still could be different due to the large differences in the levels of the presented antigen. Since minigene P18 vaccinia-infected cells expressed a much higher level of H-2Dd–P18-I10 complexes than vPE16-infected cells, we expected to see a higher number of low-avidity T cells in the minigene P18 vaccinia-immunized mice. Therefore, we compared functional and physical avidity of P18-I10-specific CD8 T cells of vPE16 and minigene P18 vaccinia-immunized mice. They were measured in parallel using a dose–response IFN-{gamma} ELISPOT assay for functional avidity (Fig. 3A) and a tetramer dissociation assay (Fig. 3B), which estimates relative TCR affinity for the P18-I10–H-2Dd complex based on the rate of tetramer dissociation under conditions that prevent rebinding. Surprisingly, no differences in avidity or affinity were observed between T cells from mice immunized with vPE16 or minigene P18 vaccinia viruses. So, the 12-fold increase of the MHC–P18-I10 complexes from 380 to 4600 does not reduce the average avidity of the T cells in minigene P18 vaccinia-immunized mice and could be a reason why we do not see an increase in immunodominance in minigene virus-immunized mice. The result was somewhat surprising, since we would expect to have more of the low- than high-avidity TCR in the naive CD8 T cell repertoire for any given pMHC target. Therefore, one would predict more recruitment and expansion of low-avidity T cells when the level of MHC–peptide complexes is increased. It is unlikely that the low level of the activation threshold, which was shown to exist in the T cells (39), is reached in the studied model since we still can detect low-avidity P18-I10-specific T cells and even grow them in vitro (40). These results rather suggest a preferential expansion of high-avidity T cells that is not affected by increased recruitment of low-avidity T cells.


Figure 3
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  		Fig. 3. Minigene P18 vaccinia and vPE16 induce P18-I10-specific T cells of the same avidity. (A) Functional avidity, IFN-{gamma} ELISPOT assay. Splenocytes of mice immunized with vPE16 or minigene P18 vaccinia were stimulated by P815 cells pulsed with different concentrations of the peptide. Values are normalized by response to maximal dose (10 µM) of P18-I10 peptide. Figure shows individual mice, n = 3 per group. Presented is a typical experiment of three with comparable results. (B) Tetramer dissociation assay. Splenocytes from immunized animals were stained with P18-I10 tetramer and then incubated for different times with 34-5-8S anti-H-2Dd mAb to prevent rebinding of tetramer with TCR, and analyzed by flow cytometry. Presented is a typical experiment of two with similar results.

 
Beneficial expansion of the high-avidity T cells may occur as a result of different mechanisms, which involve competition for the DCs, higher rate of cell death among low-avidity T cells or higher rate of proliferation of high-avidity T cells. The measurement of the level of proliferation of low- and high-avidity T cells would allow us to some extent to answer these questions.

To compare the rates of proliferation of high- and low-avidity T cells, we developed a method of direct visualization of high- and low-avidity populations based on the different dissociation rates of P18-I10-H-2Dd tetramer from the T cells of different avidity. Bound tetramer dissociates more quickly from low-avidity T cells than from high-avidity T cells. Based on this, immune cells were stained with tetramer labeled with PE; subsequently, the tetramer was allowed to dissociate from CD8+ T cells in the presence of 34-5-8S mAb to H-2Dd to block rebinding of the tetramer to the TCR (Fig. 4A). Following a 6-h incubation with the anti-MHC antibody, we observed a decrease in PE staining by 40–50%, with low-avidity cells no longer having tetramer–PE bound, but high-avidity cells retaining tetramer–PE (data not shown).


Figure 4
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  		Fig. 4. High-avidity T cells proliferate better than low-avidity ones in vivo. To visualize high- and low-avidity T cells, samples were stained with anti-CD8 mAb and P18-I10 tetramer labeled with PE, and then incubated with anti-H-2Dd 34-5-8S mAb for 6 h and finally with P18-I10 tetramer labeled with APC (A). Cells were gated on tetramer APC+CD8+ cells (B). High-avidity CD8 T cells were positive for both tetramer–PE and tetramer–APC (D) and low-avidity CD8 T cells were positive only for tetramer–APC (E). To see the difference in proliferation of high- and low-avidity T cells, mice were immunized with vPE16 in the footpad and were given BrdU in the drinking water for 4 days. Then popliteal LNs were isolated and cells were first stained for high- and low-avidity T cells as described above and for intracellular BrdU content (C). Cells were gated on tetramer APC+CD8+ cells (B), and of these, a larger fraction of tetramer PE+ tetramer APC+CD8+ cells (high-avidity T cells) have taken up of BrdU and show a higher geometric mean fluorescence than tetramer PE–tetramer APC+CD8+ cells (low-avidity T cells) (representative case, C). (F) Summary of the experiment of different mice studied as in (E) (n = 3), y-axis geometric mean fluorescence of BrdU on day 4 and 5 after immunization. Error bars are standard deviation of values for three mice per group (P < 0.001 for high- versus low-avidity T cells and P = 0.01 for low avidity versus control without BrdU). (G) Comparison of BrdU inclusion in high- versus low-avidity P18-I10-specific T cells isolated from the LNs of mice immunized with vPE16 virus or minigene P18 vaccinia virus.

 
The cells were then stained with the tetramer labeled with a different fluorochrome (APC) (Fig. 4B). CD8 T cells that retained both tetramers were considered to be of high avidity (Fig. 4D) and cells that were only APC positive (i.e. labeled in the second staining, having completely lost the tetramer–PE) were considered to be of low avidity (Fig. 4E). The percent of tetramer–PE-positive cells before incubation with anti-MHC antibody was always equal to the percent of tetramer–APC-positive cells after the incubation (data not shown). As a validation of the method, high- and low-avidity fractions of cells sorted by FACS were also shown in a functional ELISPOT assay to respond to correspondingly lower and higher concentrations of the antigen. The percent of cells responding to 1 nM peptide (high-avidity fraction) out of all specific cells responding to 1 µM peptide (a measure of the total high- and low-avidity response) was 75% for the high-avidity T cells and 25% for low-avidity ones. The ability to discriminate between high- and low-avidity CD8+ T cells specific for the same peptide–MHC complex by flow cytometry allowed us to estimate differences in the division rate in vivo. Differences in proliferation of high- and low-avidity T cells in vivo were analyzed using BrdU. BALB/c mice were immunized with minigene P18 vaccinia virus and administered BrdU in the drinking water. Draining popliteal Lymph nodes were isolated, and staining was performed 4 and 5 days after immunization for high- and low-avidity P18-I10-specific CD8 T cells with tetramer–PE and tetramer–APC, as described earlier, and for intracellular BrdU content. On day 4, we observed ~0.5% P18-I10 tetramer-positive cells out of the total CD8 T cells, whereas on day 5, ~1.5–2% of the CD8 T cells were tetramer positive. High-avidity T cells incorporated much more BrdU than did low-avidity ones on both day 4 (Fig. 4C, F and G) and day 5 (Fig. 4F). However, even though low-avidity T cells incorporated less BrdU than high-avidity ones, they still had higher levels of the mean fluorescence after staining with anti-BrdU antibody than P18-I10 tetramer-positive cells from mice that were not treated with BrdU (data not shown) indicating that low-avidity T cells do proliferate, however, with less efficiency. Later time points, 6–7 days after immunization (data not shown) demonstrated less, albeit still significant, differences in the level of the BrdU incorporation in high- and low-avidity T cells, probably due to the saturation of the BrdU incorporation. Note that the proportion of cells in each fraction incorporating BrdU is independent of any error in the measurement of the actual size of each fraction. We also found similar results with mice immunized with vPE16 instead of the minigene P18 virus, in which high-avidity T cells incorporated significantly more BrdU than low-avidity T cells (Fig. 4G). Similar differences in the rate of proliferation of high- and low-avidity T cells were observed using another approach, which involved the stimulation of T cells with different concentrations of P18-I10 peptide and subsequent staining for intracellular IFN-{gamma} and BrdU. Cells producing IFN-{gamma} after stimulation with 500 nM P18-I10 peptide incorporated less BrdU than those cells responding to stimulation with 0.5 nM of the peptide (data not shown). Even though several groups have suggested a role for T cell avidity and the rate of T cell expansion in epitope immunodominance as a potential explanation of their results, a proof of the existence of such a mechanism was not provided (41).

The differences in the rate of the expansion of high- and low-avidity T cells may partially explain the absence of a detectable increase of low-avidity T cells in the mice immunized with minigene P18 vaccinia virus and the lack of difference of the P18-I10 epitope immunodominance between mice immunized with minigene P18 vaccinia and vPE16 viruses. Such enhanced expansion of high-avidity T cells must affect the composition of the anti-viral CTL response. Some studies demonstrated enrichment of high-avidity T cell clones among T cells specific for a particular epitope, which is distinct from immunodominance of the epitope (42, 43). However, a convincing correlation between immunodominance of an epitope among all the epitopes of a protein or whole virus and avidity of epitope-specific T cells has not yet been demonstrated.

One of the major problems that we encountered while setting up the experimental model was a difficulty in accurately comparing the functional avidity of the T cells specific to the different epitopes due to the varying MHC-binding properties of the different peptides. As an alternative, we propose a model in which we would be able to use just one epitope while having the level of its immunodominance vary; and in this setting, we would maintain the identity and the level of the peptide–MHC complexes constant. If immunodominance depends on avidity and therefore, among other things, T cell repertoire, then we would predict it to vary among outbred humans with the same HLA molecule but different TCR genes. To test this prediction in mice, we compared congenic strains of mice that share the same MHC but differ in other genes that affect their TCR repertoire, including the TCR genes themselves as well as other genes affecting positive and negative selection. Such mice should serve as a model of an outbred population of individuals sharing their MHC genes. We had found that immunodominance differs significantly between MHC-congenic mice with different genetic background, which allowed us to pursue this approach.

MHC-congenic mice have differing naive CD8 T cell repertoires due to the effects of non-MHC gene-derived peptides on positive and negative selection in the thymus, as well as potential differences in available TCR genes. These differences could be partially responsible for the differences in the immunodominance. Major differences in Vß gene usage were observed among naive CD8+ sub-populations of H-2Dd congenic mouse strains BALB/cJ, B10.D2/nsn, DBA/2J, LG/J and B6.C-H2d especially in their use of Vß3, Vß5.1 and 5.2, Vß6, Vß7, Vß9, Vß10b, Vß11 and Vß12 (data not shown).

BALB/cJ, B10.D2/nsn, DBA/2J, LG/J and B6.C-H2d mice (all expressing H-2Dd) were immunized with recombinant vaccinia virus vPE16, expressing P18-I10. The number of antigen-specific cells was assessed directly ex vivo 2 weeks after immunization by ELISPOT for IFN-{gamma}-producing cells and by P18-I10-H-2Dd tetramer. All groups of mice responded to the P18-I10 antigen, although the magnitude of the CD8 T cell response differed significantly (Supplementary Figure 2A and B, available at International Immunology Online). We also observed a significant variance in the Vß7, Vß13, Vß9, Vß8.3 and Vß8.1 usage of P18-I10-specific cells (data not shown). Although other potential differences in the immune response, such as the innate or the humoral immune response, may affect absolute viral load and the level of CD8 T cell response, we normalized for that effect when we calculated the immunodominance ratio of the P18-I10 response to the response against the whole virus. The calculation of the immunodominance in these mice was particularly necessary, since mice with different genetic backgrounds had different levels of immune response to the vaccinia virus.

The lowest and highest levels of ID of the P18-I10 epitope relative to the whole vaccinia response were observed in B10.D2 (14%) and in LG mice (43%), respectively. To examine the possibility of a correlation between avidity and immunodominance, we measured avidity of P18-I10-specific T cells. The proportion of high-avidity cells was determined based on the number of cells responding to a low concentration (1 nM) of the peptide compared with the number of cells able to respond to a higher concentration (10 µM). As predicted from our data in BALB/c above, the level of immunodominance strongly correlated with the functional avidity of the CD8+ T cells (Fig. 5A and B), providing support for our working hypothesis.


Figure 5
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  		Fig. 5. Mice with different genetic backgrounds have different levels of immunodominance of the P18-I10 epitope, and immunodominance of P18-I10 in these mice correlates with the avidity of P18-I10-specific T cell (A and B). Immunodominance of the P18-I10 epitope and avidity of P18-I10-specific T cells differ significantly in strains of mice with different genetic backgrounds (A) and individual mice (B) that demonstrate clustering accordingly to their background. The level of immunodominance was calculated as the percent of total anti-viral response that is P18-I10 specific. Percent of high-avidity T cells is the percent of T cells responding to 1 nM out of the cells responding to 10 µM. The percent of P18-I10 immunodominance correlated with the percent of high-avidity P18-I10-specific T cells [R = 0.91, P = 0.03 for (A) and P = 0.0005 for (B)]. (C) The level of ID of the P18-I10 did not correlate with the magnitude of immune response in these mice by tetramer staining. (D) The level of immunodominance also did not correlate with the ability of the CD8+ T cells in these mice to expand on anti-CD3 stimulation.

 
Besides measuring the ID and functional avidity in these mice, we also studied the ability of CD8+ T cells to expand after activation. The ability of T cells to expand after stimulation with anti-CD3 mAb was measured by labeling cells with CFSE and subsequent analysis of the numbers of the CD8 T cell divisions. As seen in Fig. 5(C), the level of P18-I10 immunodominance in these mice did not correlate with the magnitude of the immune response measured by tetramer and IFN-{gamma} ELISPOT (data not shown) or with the ability of T cells to expand after anti-CD3 stimulation (Fig. 5D).

These results indicate an important role of high-avidity T cells in the development of the immune response and immunodominance to a particular epitope and a lack of dependence on the number of MHC–peptide complexes once a certain threshold is reached. Since selective expansion of high-avidity T cells was not due to low levels of the antigen, we hypothesized that high-avidity T cells in our system might undergo faster expansion than low-avidity T cells due to a higher rate of proliferation.


   Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
Supplementary data
 References
 
Our findings, in conjunction with those of others (42, 43) demonstrate that avidity of the T cells plays an important role in the shaping of the immune response. Our observation also significantly extends the idea of the influence of high-avidity T cells on the immune response from the dominance of single high-avidity T cell clones within the response to a particular single epitope to the dominance among the groups of high-avidity T cell clones to the different epitopes, the phenomenon that we detect as immunodominance.

Another process that affects participation of low-avidity T cells in the immune response is the T cell activation threshold level of the peptide–MHC complexes, studied previously in detail (39). We note that the threshold level of activation for the lowest avidity P18-I10 epitope-specific cells is far higher than in any experiments demonstrated here. Previous studies of P18-I10 epitope demonstrated that some of the lowest avidity T cell clones generated to P18-I10 epitope had the peak response at as high as 10 µM concentration of the peptide (40, 44), which corresponds to ~15 000 molecules per cell, a much higher level than is expressed in minigene P18 vaccinia-infected cells. On the other hand, a continuum of avidities was observed when T cells were stimulated with intermediate concentrations of peptide (40, 44), so it is still somewhat surprising that no difference in avidity was seen between immunization with vPE16 and immunization with the minigene P18 virus. Even without counting the numbers of high- and low-avidity T cells, the nearly superimposable dose–response curves and tetramer dissociation kinetics (Fig. 3A and B) indicate that there was little difference in the avidity distribution among T cells from mice immunized with vPE16 versus the minigene P18 virus.

In contrast to the passive threshold for T cell activation, the differential level of expansion of high- and low-avidity T cells demonstrated in our study actively skews the repertoire towards high-avidity T cells. Absence of a significant impact of the epitope density of P18-I10–MHC complexes on the surface of APCs after certain levels are reached may imply an important role of CD8 T cell precursors in the development of the immunodominance. The studies of the role of the CD8 T cell repertoire already showed that frequency of CD8 T cell precursors affects the level of the immunodominance. An increase in the numbers of T cell precursors was shown to increase the immunodominance and the practical application based on this principle—the heterologous prime-boost vaccination strategy—becomes increasingly important in vaccine design. However, since the T cell repertoire theoretically can have as many as 1014 different TCR, the question that still remains is why some epitopes elicit more specific T cell precursors than the others. It is known that the T cell repertoire is shaped by positive and negative selection in the thymus. Therefore, it is logical to suspect that either of these processes may affect the immunodominance as well. There is also direct evidence demonstrating a possible influence of negative selection on epitope dominance, creating so-called ‘holes’ in the repertoire. For instance, ovalbumin-transgenic C57BL/6 mice (45) are completely tolerant to SIINFEKL (the dominant epitope of the ovalbumin). However, some studies suggest that T cell repertoire practically far exceeds the possible overlap of self- and foreign epitopes for these holes in the repertoire to significantly affect the recognition of the majority of epitopes. The role of positive selection in the shaping of the T cell repertoire is controversial (46, 47). However, direct experiments with knockout mice (47) suggest rather minimal participation of positive selection in the development of immunodominance. Therefore, the reason for higher frequency of T cell precursors to particular epitopes is mostly unclear and may involve yet unknown mechanisms. The answer could be buried in the ability of the epitope itself to interact with less variable regions of the TCR while in contact. For instance, peptide presented by an MHC molecule mostly contacts the CDR3 region of the TCR (48), the most variable part of the molecule. Thus, one would suggest that the peptides that at least partially interact with other less variable regions of the TCR would be able to recruit a larger fraction of T cell clones. Despite these speculations, the theoretical distribution of T cell precursors specific to the different epitopes still might resemble a Gaussian distribution, which is in contrast to the observed spikes of the response to just few of the antigenic determinants. Our findings may help to understand the following reasons for this: different epitopes may have differences in average avidity of T cell precursors and the resulting initial even small differences in the average rate of proliferation of the T cells specific for the different epitopes will result in a large difference in the final level of epitope-specific cells after multiple cell divisions. For example, a 20% higher average division rate of the cells specific for one epitope compared with another one will make the former T cells 15-fold more abundant after 15 divisions. Thus, we may speculate that the most noticeable effect of the immunodominanace response to the dominant epitopes that exceeds by many fold the response to other epitopes indeed could be a result of such differential expansion of the T cells specific for the different epitopes. Even though our findings strongly suggest this interpretation, further direct evidence still may be required to confirm this conclusion.

Although our observations do not provide a quantitative estimate of the degree of the influence of T cell avidity on the level of immunodominance, they still offer a tool for exploring some answers to questions of immunogenicity of different epitopes. For instance, it is known that some non-immunogenic self-epitopes may induce a very strong immune response after just one amino acid substitution (47, 49). Also, we know that low-avidity T cells to one epitope function with high avidity with another epitope and vice versa. So, based on our observations, we may suggest that available T cell precursors had higher avidity to the modified peptide than to the wild type and therefore had better potential for proliferation, which in turn increased the immunodominance of the epitope.

The biological significance of the immunodominance is still an enigma. Based on our findings, we may support an idea that immunodominance possibly plays a role in the protection against self-reactivity. It is known that T cells with high-avidity TCR towards self have a very small chance to escape negative selection in the thymus. Therefore, if a high-avidity T cell recognizes an antigen–MHC complex on the surface of the DC, there is a low chance that the antigen is self. In contrast, antigen recognized by T cells on DCs with low affinity could potentially be self (50), as such T cells may escape negative selection in the thymus. Therefore, expansion of high-avidity T cells may result in much lower risk for the development of autoimmunity than expansion of low-avidity T cells. Here we demonstrated that more rapid expansion of high-avidity T cells compared with lower avidity ones is one of the mechanisms responsible for the development of immunodominance. Therefore, it is possible that immunodominance could be the result of a mechanism preventing the development of autoimmunity. That algorithm seems to be especially important if one remembers that the immune response develops in a situation where professional presenting cells present both self- and foreign antigens. These findings also demonstrate yet another important role of T cell avidity and may contribute to design of more effective vaccines for infectious diseases and cancer (40-44, 51-54).


   Supplementary data
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
References
 
Supplementary figures 1 and 2 are available at International Immunology Online.


   Acknowledgements
 
We thank Drs. James Gibbs, Jonathan Yewdell, and Jack Bennink for a gift of the minigene P18 vaccinia virus, Drs. Bernard Moss and Patricia Earl for a gift of vPE16 and vSC8, Susan Sharrow and Larry Granger for cell sorting and Roxana Moslehi for consultation in the analysis of the data. We thank Drs. Ronald Germain, Susan Gagnon, and Jonathan Yewdell for critically reviewing this manuscript. This work was carried out with the support of the intramural program of the Center for Cancer Research, National Cancer Institute and NIH.


   Abbreviations
 
APC, antigen-presenting cell
BrdU, 5-bromo-2-deoxyuridine
CFSE, carboxyfluorescein succinimidyl ester
DC, dendritic cell
ER, endoplasmic reticulum
MFI, mean fluorescence intensity
NIAID, National Institute of Allergy and Infectious Diseases
PFU, Plaque-forming units

   Notes
 
Transmitting editor: T. Sasazuki

Received 20 September 2006, accepted 24 January 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 

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