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* Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia;
Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, TN 38105; and
Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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by immunodominant Ag-specific CTL (6), and the suppression of specific CTL responses by other, dominant responses (7, 8). The ability of a specific CTL response to suppress another has been termed immunodomination. This can be clearly observed where immunodominant memory CTL recalled during a secondary response can suppress subdominant primary responses (2, 9). The ability of CTL responses to dominate others has been attributed to T cell competition for access to Ag on the same APC (7, 8, 10). Demonstration of immunodomination is dependent on transfer of high numbers of naive TCR transgenic (Tg)3 CTL (7, 8). However, although it has been shown that while transfer of high numbers of naive CTL can inhibit the host responses of the same specificity (7, 8), there is less impact on CTL of other specificities (7, 11). The conclusion from such studies is that generation of different specific CTL responses is independently regulated after infection (7).
Respiratory infection of C57BL/6 (H2b) mice with influenza A virus generates CTL responses directed against a total of 10 different determinants (12), with the most prominent derived from the nucleoprotein (NP366–374, H2Db) (13, 14), the acidic polymerase (PA224–233, H-2Db) (15), and the basic polymerase subunit 1 (PB1703–711, H2Kb) (16). Although these CTL responses are codominant during the primary response to infection (15, 16, 17), the DbNP366-specific CTL population dominates the response after secondary challenge, constituting up to 80% of the influenza-specific CD8+ population (16, 18, 19). Immunodominance of the DbNP366-specific CTL response in the secondary response has been attributed recently to a broad spectrum of cellular Ag presentation (20), increased level of NP366 epitope presentation compared with the other epitopes (21), and the possibility the DbNP366 response can immunodominate the DbPA224-specific response in the secondary response (22). However, it has been demonstrated that the primary DbPA224 response is not affected after transfer of DbNP366-specific CTL (22, 23). Furthermore, in the absence of a secondary DbNP366 response, the DbPA224-specific CTL response fails to compensate to any significant extent (9, 24). These studies imply that immunodomination by DbNP366 may in fact play a minor role in determining the magnitude of the DbPA224-specific response. However, concurrent examination of subdominant DbPB1-F262–70-, KbPB1703-, and KbNS2114-specific responses showed an increase in magnitude in the absence of DbNP366- and DbPA224-specific responses (9, 24). The conclusion from these studies was that subdominant CTL responses compensate for loss of immunodominant responses after influenza A virus infection. Given the discrepancy in the literature, further studies are required to help determine the consequence of CTL immunodomination on Ag-specific hierarchies established after influenza A virus infection.
To further examine the potential for immunodomination in the influenza A-specific CTL response after infection, primary and secondary DbNP366, DbPA224, and KbPB1703 CTL responses were analyzed after infection with an influenza A virus containing the immunogenic OVA peptide (OVA257; amino acid sequence SIINFEKL, presented by H2Kb) within the stalk of influenza virus neuraminidase (NA) protein. The presence of an immunodominant KbOVA257-specific CTL response resulted in a trend for diminished CTL responses to the native influenza A virus epitopes, although this result was variable. Interestingly, the presence of KbOVA257-specific CTL responses could reproducibly suppress the DbPA224- and KbPB1703-specific CTL responses when Ag was limiting during a nonproductive infection. Moreover, transfer of OT-I TCR Tg CTL specific for the KbOVA257 epitope into naive mice resulted in diminished CTL responses to DbNP366, DbPA224, and KbPB1703 after infection. Importantly, this effect could not be rescued for DbPA224- and KbPB1703-specific CTL responses by increasing the level of epitope presentation. Overall, these data suggest that in cases in which either viral Ag is limiting, or the T cell precursor frequency for particular epitopes is high, CTL immunodomination of other specificities is apparent.
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Materials and Methods |
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Female C57BL/6J (B6, H-2b) Thy-1.2+ and Tg B6-OT-I Thy-1.1+ mice were bred in the animal facility at the Department of Microbiology and Immunology at University of Melbourne and were held under specific pathogen-free conditions. Naive mice at least 6 wk of age were lightly anesthetized by inhalation of (methoxyfluorane) penthrane and infected intranasally (i.n.) with 104 PFU of A/HKx31 strains of virus in 30 µl. Mice for secondary challenge experiments were primed by i.p. injection with 1.5 x 107 PFU of the A/PR8 viruses at least 6 wk previously. The A/HKx31 and A/PR8 influenza A viruses differ in their surface hemagglutinin and NA, but share the PR8 internal proteins (NP, NS1, NS2, M, PA, PB1, and PB2). At the time of tissue sampling, mice were anesthetized and exsanguinated by section of the axillary artery. The inflammatory cell population obtained by bronchoalveolar lavage (BAL) was incubated on plastic for 1 h at 37°C to remove adherent macrophages (25). In one experiment, lymphocytes were isolated from perfused, minced lung tissue by collagenase digestion (19). Single-cell suspensions of spleen were enriched for CD8+ T cells by incubation on plastic tissue culture plates precoated with 200 µg/ml anti-IgG and anti-IgM Abs (Australian Laboratory Services) for 1 h at 37°C, 5% CO2.
Generation and titration of recombinant viruses
PCR primers encoding for the OVA257–264 SIINFEKL peptide and part of the NA from A/HKx31 or A/PR8 (sequences are available upon request) were used with NA-specific primers (26) to amplify the NA segment from either A/HKx31 or A/PR8. The PCR products were digested with BsmBI and ligated into pHW2000 (26). The recombinant A/HKx31 and A/PR8 viruses (in this study referred to as HK-OVA and PR8-OVA, respectively) were rescued after transfection of mixed cultures of 293T and Madin-Darby canine kidney (MDCK) cells using the eight-plasmid reverse genetics system described by Hoffmann et al. (27). Recombinant influenza A viruses were grown at 33°C for 2 days in the allantoic cavity of 10-day embryonated hen eggs, then incubated at 4°C overnight before the allantoic fluid was harvested and clarified by centrifugation at 3000 x g for 10 min. The virus stocks used for infection were grown in eggs. Viral titers were determined from allantoic fluid (or lung homogenates from infected mice) by plaque assay on confluent MDCK cell monolayers (28).
Peptide stimulation and intracellular cytokine staining
Enriched spleen- and macrophage-depleted BAL lymphocyte populations were cultured for 5 h at 37°C in 96-well round-bottom plates at 
0.5–2 x 106 cells/well in complete RPMI 1640 containing 10% FCS, 10 U/ml human rIL-2, and 5 µg/ml GolgiPlug (BD Biosciences), with or without 1 µM NP366–374 (amino acid sequence ASNENMETM), PA224–236 (amino acid sequence SSLENFRAYV), PB1703–711 (amino acid sequence SSYRRPVGI), or OVA257–264 (amino acid sequence SIINFEKL) peptides (Auspep). The cells were then washed with PBS (containing 0.1% BSA and 0.02% sodium azide), stained with anti-mouse CD8
-PerCPCy5.5 (BD Pharmingen) for 30 min on ice, permeabilized by paraformaldehyde fixation using the BD Cytofix/Cytoperm Kit (BD Biosciences), and stained for intracellular cytokine production using anti-mouse IFN- FITC (clone XMG1.2; BD Pharmingen) and anti-TNF- allophycoerythrin (clone MP6-XT22; BD Pharmingen). The lymphocytes were then washed and analyzed on a FACSCalibur, and analysis was performed using CellQuestPro software (BD Biosciences). In each assay, any cytokine-positive cells isolated from wells with no peptide were subtracted from the percentage of cytokine-positive cells incubated with peptide to yield the final value.
CD8+ T cell lines and CTL assay
Splenocytes from naive B6 mice were resuspended (108 cells/ml) in HBSS, incubated with 1 µM peptide for 1 h at 37°C (15), irradiated (3000 rad), washed twice in HBSS, and mixed (3 x 107 cells) with equal numbers of splenocytes from B6 mice that had been infected i.n. 10 days previously with 104 PFU of the HK virus. They were then resuspended in 40 ml of complete RPMI 1640 (10% FCS/penicillin-streptomycin/glutamine/5 x 10–5 M 2-ME) and cultured for 5 days at 37°C, 5% CO2. After 5 days, a CTL assay was performed. The target EL4 cells used for the CTL assay were either infected with 10 multiplicity of infection of PR8-OVA or HK-OVA (1 h in 300 µl of HBSS, then 2 h in RPMI 1640) or pulsed with 1 µM peptide, then labeled with 300 µCi of 51Cr (Amersham Biosciences) for 1 h at 37°C. Effector T cells were serially diluted across a range of E:T ratios in 96 round-bottom plates. The percentage of specific lysis at 6 h was calculated as 100 x (51Cr release from targets with effectors – 51Cr release from targets alone)/(51Cr release from targets with 1% Triton X-100 – 51Cr release from targets alone). The level of 51Cr release from targets alone did not exceed 10% of the total 51Cr release from targets with 1% Triton X-100.
Adoptive transfer experiments
Naive splenocytes were obtained from B6-OT-I-CD45.1+ mice (29) and enriched for CD8+ T cells by depletion after incubation on plastic tissue culture plates precoated with 200 µg/ml anti-IgG and anti-IgM Abs for 1 h at 37°C, 5% CO2, before staining 1 x 107 cells/ml with 5 µM fluorescent dye CFSE (Sigma-Aldrich), according to Turner et al. (23). The labeled cells were transferred i.v. into naive B6 CD45.1– mice. Before transfer, the CD8+ T cell population displayed the naïve CD69lowCD62LhighCD44high phenotype (data not shown). Recipient mice were infected i.n. with 1 x 104 PFU of HK-OVA 24 h after cell transfer and sampled after an additional 8 days.
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Results |
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A reverse genetics strategy (27) was used to insert the OVA257–264 peptide (SIINFEKL) presented by H2-Kb into the NA stalk of the A/PR8 (H1N1) and A/HKx31 (H3N2) influenza A viruses. The ability of peptide-stimulated CTL lines to kill either PR8-OVA or HK-OVA virus (data not shown)-infected EL4 (DbKb) cells established that infection with either virus induced the expression of the KbOVA257 epitope (
; Fig. 1A). The level of KbOVA257-specific 51Cr release was broadly equivalent to the killing activity found for the native DbNP366 after PR8 infection (compare ; Fig. 1, A and B). Furthermore, the presence of OVA257 in the viral NA neither changed the sensitivity of the infected EL4 cells to DbNP366-specific CTL lysis (Fig. 1B, compare • and ), nor modified the apparent lack of expression of the DbPA224 epitope (Fig. 1C, compare • and ). Both the PR8-OVA and HK-OVA viruses thus induce the expression of KbOVA257 without obviously altering the normal MHC class I-restricted presentation profile characteristic of infection with these influenza A viruses.
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Previous reports have demonstrated that insertion of short peptides into the NA of influenza A virus strain, A/WSN/33, can result in attenuation of in vivo virus growth after infection (30, 31). To determine whether the presence of the OVA257 epitope in the NA of HK-OVA resulted in attenuation of the virus, mice were infected i.n. with equivalent doses containing 104 PFU of either the HK or HK-OVA viruses. Virus lung titers were determined by plaque assay at intervals after respiratory exposure. At all time points assayed, the HK-OVA viral titers were significantly lower than in mice given an equivalent dose of HK virus (p < 0.01 at day 3 postinfection; p < 0.03 at days 5 and 7 postinfection) (Fig. 2A). The lower HK-OVA viral titer at day 3 was unlikely to be due to a KbOVA257-specific response, as infection of MHC-mismatched BALB/c mice (H-2d) with the HK-OVA virus also resulted in lower peak viral titers at day 3 compared with the wild-type HK virus (Fig. 2B). Interestingly, despite the HK-OVA virus growing to a lower peak viral titer, it was cleared at the same time as the wild-type HK virus (Fig. 2A). Therefore, in agreement with previous studies (30, 31), it seems that insertion of the OVA257 peptide into NA resulted in attenuated viral growth in vivo. Such attenuation may have relevance for i.n. infection where productive infection by the HK-OVA virus may result in less Ag, and therefore not induce robust CTL responses.
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Given the observed attenuation of the HK-OVA virus, it was important to determine whether infection with HK-OVA would result in a decreased magnitude of CTL responses compared with HK infection. To determine both the impact of the attenuation and the impact of OVA257 presentation on the size of responses directed toward DbNP366, DbPA224, and KbPB1703, B6 mice were infected i.n. with 104 PFU of the HK or HK-OVA viruses and sampled 10 days later. The lymphocytes obtained from spleen or by BAL were stimulated with 1 µM NP366 (Fig. 3, A and E), PA224 (Fig. 3, B and F), PB1703 (Fig. 3, C and G), or OVA257 (Fig. 3, D and H) peptides in the presence of brefeldin A, then stained subsequently with IFN-- and TNF--specific mAbs. Representative staining profiles are shown for the spleen (Fig. 3, A–H). In agreement with previous studies (16, 17), the majority of DbPA224-specific CTL are positive for both IFN- and TNF- after peptide stimulation (Fig. 3, B and F), while DbNP366-specific/TNF-+ CTL are only a subset of the IFN-+ CTL (Fig. 3, A and E). The cytokine profile of KbOVA257-specific CTL (Fig. 3H) was more similar to that of DbNP366-specific CTL with TNF-+ CTL, a subset of the IFN-+ CTL (compare Fig. 3, E and H). Importantly, there was no evidence that OVA from egg allantoic fluid used to grow the virus stocks induces a KbOVA257-specific response when the virus is administered i.n. (Fig. 3D).
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+ populations generated following primary challenge showed that concurrent expansion of the CD8+KbOVA257+-specific set did not significantly diminish the size of the DbNP366+ CTL response recovered from the spleen (Fig. 3I) or the infected lung (Fig. 3K). There was a trend for diminished DbPA224- and KbPB1703-specific responses in the spleen and lungs of mice that were infected with HK-OVA (Fig. 3, I and K). Although these diminished responses were variable and not significantly different in the spleen, in the lung there were 50% fewer KbPB1703-specific T cells following HK-OVA infection compared with wild-type virus infection (Fig. 3K; p < 0.05). Importantly, the responses to DbNP366 and KbOVA257 were essentially comparable in magnitude (Fig. 3, I and K). By this criterion, KbOVA257 can be regarded as a prominent epitope in the primary response to the HK-OVA virus. Summing the values for the four populations of CD8+ T cells indicated that, despite the apparent attenuation of HK-OVA, the total numbers of responders in either the BAL or spleen were, in fact, similar after infection with either HK or HK-OVA (Fig. 3, J and L). This is most likely due to the presence of the KbOVA257-specific response making up for the diminished DbPA224 and KbPB1703 responses. Effect of KbOVA257 on the influenza A virus-specific CTL response following secondary infection
The consequences of OVA257 expression for secondary challenge were next analyzed using mice that were primed i.p. with PR8-OVA, then given either the HK (Fig. 4, A–D) or the HK-OVA (Fig. 4, E–H) viruses i.n. 6 wk later. Lymphocytes from both the spleen and BAL were stimulated with the various peptides, and the production of IFN- and TNF- was determined. Representative staining profiles are shown for the spleen (Fig. 4, A–H). After challenge with HK-OVA, the proportion of CD8+ CTL specific for either KbOVA257 or DbNP366 was similar (Fig. 4, compare E and H). Furthermore, the proportion of TNF-+ of IFN-+ KbOVA257-specific CTL was again similar to that observed for the DbNP366-specific set as seen after primary infection (Fig. 4, A, E, and H). Memory KbOVA257-specific CTL observed after HK challenge were largely IFN-+/TNF-+ (Fig. 4D), as has been described for other influenza-specific memory CTL populations (17). Memory KbOVA257-specific CTL could be found in the spleens and lungs of HK-infected animals, but showed no evidence of clonal expansion (Fig. 4, I and K, and data not shown), supporting the notion that specific Ag is required for expansion of memory CTL (23, 32). As in the primary response, there was a trend for diminished DbPA224- and KbPB1703-specific CTL responses in the presence of an immunodominant KbOVA257 response, although these changes were not significant (Fig. 4, I and K). Although the presence of a large recall KbOVA257 response resulted in a trend toward a greater total response in the spleen and infected lung, compared with HK secondary challenge, this difference was also not significant (Fig. 4, J and L). Overall, the pattern following both primary and secondary challenge reflected that the presence of the prominent secondary KbOVA257 response caused no substantial change in the total magnitude of the CD8+ T cell response.
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The earlier results suggested that the presence of the immunodominant OVA257-specific CTL response resulted in diminished DbPA224- and KbPB1703-specific responses, although these results demonstrated a degree of variability. Importantly, despite the in vivo attenuation of the HK-OVA virus, the total specific CTL response was similar for both HK and HK-OVA viruses. Therefore, over the course of a replicative infection, enough Ag may be present for the optimal expansion of influenza A virus-specific population, reducing the impact of the OVA257-specific response.
It is possible that the immunodomination of KbOVA257 might be more obvious if viral dose, and therefore Ag load, was equalized for both viruses. Intraperitoneal infection with influenza A virus does not result in the production of mature virus due to the lack of a tissue-specific enzymatic cleavage of hemagglutinin required for replicative infection. Therefore, i.p. challenge results in nonreplicative infection, and consequently, the effective Ag dose following influenza virus challenge by a nonrespiratory route may be considered a direct reflection of the amount of virus (whether infectious or defective) in the input inoculum. Mice were primed i.p. with PR8-OVA, rested for 6 wk, then challenged i.p. with 1.5 x 107 PFU of HK or HK-OVA (Fig. 5). In mice given HK-OVA, the magnitude of CD8+DbNP366-, CD8+DbPA224-, and CD8+KbPB1703-specific responses was significantly diminished when compared with mice given the HK-OVA virus (Fig. 5, A and B).
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Consequences of adding Tg OVA257-specific T cells to the response
A key characteristic of CD8+ T cell memory is the increased size of the responder population (33), which is generally considered to be at least 100-fold higher than the naive T cell precursor frequency (34, 35). The secondary challenge experiments shown to date (Figs. 4 and 5) analyzed the nature of the recall response for a spectrum of memory T cell populations expanding in the presence, or absence, of a primed KbOVA257-specific set. Importantly, the immunodominant OVA257-specific response only reproducibly diminished the host response to influenza A virus determinants when Ag was limiting. The availability of the HK-OVA virus and congenic TCR Tg OT-I mice, specific for KbOVA257, allows analysis of the naive response to influenza epitopes when a naive TCR Tg population is both present and stimulated during the course of respiratory infection.
Conventional, naive Ly-5.2+CD45.1–, B6 mice were injected with 106 naive (CD62LhighCD44low), CFSE-labeled, Ly-5.1+CD45.1+ OT-I T cells. Half of these mice were left unchallenged, while the remainder (along with OT-I– B6 controls) were infected i.n. with 104 PFU of HK-OVA 1 day after transfer. The substantial response to KbOVA257 in the normal B6 mice was, of course, mediated by CD45.1– cells (Fig. 6, A and B), while almost all of the KbOVA257-specific set detected in the spleen or lung of those given the OT-I cells originated from the transferred CD45.1+ population (Fig. 6, C and D, compare upper left and upper right panels).
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– set (Fig. 6D) that is present at much higher relative prevalence than in the spleen (Fig. 6C). This suggests there is recruitment of noncytokine-producing epitope-specific T cells that do not make cytokine to the site of inflammation. The total number of CD8+IFN-+ Ag-specific T cells (including the Ly-5.1+CD45.1+ OT-I CTL) in the spleen (Fig. 7A) was unchanged. Interestingly, in the lungs of mice that received the OT-I T cells, there was a significant increase in the total number of IFN-+ CD8+ T cells (Fig. 7B).
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Transfer of OT-I T cells can suppress not only the host OVA257-specific response, but also the host response to influenza A virus-specific determinants after HK-OVA infection (Fig. 6). In contrast, a similar analysis using lymphocytic choriomeningitis virus infection demonstrated that transfer of P14 TCR Tg T cells could diminish the host response to the same specificity, but left other specific responses unaffected (7). Moreover, immunodomination observed in this study was only apparent when large numbers of P14 TCR Tg cells were transferred. Therefore, the results we observed may have been due to the large number of transferred OT-I TCR Tg cells.
To determine whether lower numbers of transferred OT-I cells would impact less on the host response to influenza A virus infection, naive CD45.1– B6 mice received either 1 x 104 (Fig. 8, Low OT-I) or 1 x 107 (Fig. 8, Hi OT-I) naive OT-I T cells. Groups of mice were infected with HK-OVA, as previously described, and the host (CD45.1–) CTL response was determined by IFN- production after stimulation with the appropriate peptide. In the spleen, the KbPB1703 and KbOVA257 host response was diminished after transfer of both high and low numbers of OT-I cells (Fig. 8, A and C). However, when the lymphocyte population isolated from the lung by BAL was analyzed, there was clear evidence of diminished host responses to DbNP366, DbPA224, KbPB1703, and KbOVA257 with transfer of both high and low numbers of OT-I cells (Fig. 8, B and D). Again, there was a hierarchy with the DbNP366 response less affected by the transfer of OT-I T cells compared with the other responses (Fig. 8). Overall, this suggests that even low numbers of OT-I precursors can inhibit host responses after infection, particularly at the site of infection.
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It has been proposed that immunodomination of the host responses by TCR Tg T cell transfer is a result of competition for Ag on APCs (7, 8). To determine whether OT-I domination of influenza A virus host responses could be overcome with more viral Ag, mice that had received 1 x 107 OT-I cells were infected with either HK, or HK-OVA, or coinfected with both HK and HK-OVA viruses (Fig. 9). The Ag-specific lymphocyte populations from the spleen (Fig. 9, A and C) and infected lungs (Fig. 9, B and D) were measured by IFN- production, as previously described.
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Discussion |
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A recent study demonstrated that the magnitude of the DbNP366-specific response did not alter after primary infection with the influenza A virus strain, A/WSN, expressing the KbOVA257 epitope (32). This is in agreement with the notion that immunodominance hierarchies established after primary infection are largely independent of each other (7). A similar conclusion was reached after analysis of the CTL responses generated after primary infection of mice with recombinant influenza A viruses lacking the immunodominant DbNP366 and DbPA224 epitopes. In this instance, there was little increase in the magnitude of subdominant CTL responses (9, 24), suggesting that the CTL responses generated were independent of each other. Although primary influenza A virus immunodominance hierarchies are established independently of each other, closer analysis of the secondary CTL response demonstrated that the magnitude of minor CTL responses increased significantly in the absence of the immunodominant DbNP366- and DbPA224-specific CTL responses. This suggests that immunodomination may play a role in determining CTL immunodominance hierarchies after influenza A virus infection (9), especially when there are large numbers of competing T cells.
It was possible to reproducibly decrease the magnitude of normal virus-specific CD8+ T cell responses under conditions where either Ag was limiting, due to nonproductive replication, or where there was excess KbOVA257-specific naive CTL precursors. In this study, diminished responses to KbPB1703, and to a lesser extent, DbPA224 and DbNP366 were observed. A possible explanation for this hierarchy might be that available Ag on APCs was more limiting for PB1703 and PA224 compared with the NP366. We have demonstrated previously that there is a greater abundance of NP and NA (where the OVA257 epitope is inserted) mRNA, compared with the PA and presumably PB1 (21). If this is taken as a correlate of protein expression, then there would be less PB1703 and PA224 presented on the surface of APCs. The PB1703 epitope would also have to compete with the OVA257 epitope for binding sites on available Kb MHC class I molecules. The notion that Ag presentation by APCs can influence immunodominance hierarchies has been proposed by numerous studies (2, 7, 8, 20). The limiting nature of Ag presented by APC can result in competition for both Ag/MHC complexes by T cells of the same specificity (8), and/or the available space on the APC for CTL of different specificities (7). Importantly, the constraint on Ag presentation for the APC does not stem from the killing of APCs by specific CTL (8).
In the case of a nonproductive influenza A virus challenge, in which a low influenza A virus dose is used, it would be expected that dendritic cells that are nonproductively infected travel via afferent lymph (or blood) to the regional lymph nodes and spleen, where they stimulate naive or memory T cell populations (20, 36). Use of the HK-OVA virus results in the recruitment of another, immunodominant T cell response specific for OVA257, leading to competition for available space and/or peptide/MHC complexes on the APC surface. There were varying levels of immunodominance effects, with the KbPB1703- and DbPA224-specific responses most affected, followed by DbNP366-specific response. It is tempting to speculate that this may relate to different levels of presentation of the various peptide/MHC complexes on the surface of APCs. It has been demonstrated that the spectrum of Ag presentation is much more limited for PA224 compared with NP366 (20).
Similarly, when large numbers of naive OT-I KbOVA257-specific T cells were introduced into the responder milieu, a similar hierarchy of immunodomination was observed with the host KbOVA257 most affected, followed by KbPB1703, DbPA224, and DbNP366. This contrasts with earlier experiments in which transfer of large numbers of Tg T cells resulted in diminished host responses only of the same specificity (7, 8). The suggestion was that responder T cells compete for sites on APCs in an epitope-specific fashion (7, 8). The difference in susceptibility to immunodomination between DbPA224 and DbNP366 may reflect peptide affinity for MHC. This seems unlikely as measurement of pMHC stability as a correlate for peptide affinity showed that DbPA224 has higher affinity than DbNP366 for H2Db (22) (N. La Gruta, P. C. Doherty, and S. J. Turner, submitted for publication). Therefore, the increased susceptibility of DbPA224 responses to immunodomination does not reflect peptide affinity for MHC. This supports earlier findings suggesting that peptide affinity plays only a minor role in determining immunodominance hierarchies (2, 22). Perhaps the fact that clonal expansion of the KbPB1703-specific T cells also seems to be relatively diminished is fortuitous, and simply reflects that this was the smallest response analyzed. An alternative explanation is that that there is some level of competition for binding of both OVA257 and PB1703 to the available Kb molecules within the APC population. Such competition might reflect differences in either peptide affinity for the Kb molecule, differences in efficiency of epitope processing and presentation, or both (2). Combined with the increased OT-I precursor frequency, this results in an almost complete abrogation of the PB1703-specific response.
Importantly, unlike earlier studies (7), transfer of small numbers of OT-I CTL still significantly diminished the host responses to KbOVA257, KbPB1703, DbPA224, and DbNP366. This, together with the fact that the responses to the KbPB1703 and DbPA224 could not be rescued by coinfection with HK virus, demonstrates that the OT-I CTL are potent inhibitors of subdominant CTL responses. All of the OT-I cells in mice that received low numbers of Tg cells underwent >8 cellular divisions, while this was not the case in mice that had received high numbers of OT-Is (data not shown). Despite this, the overall magnitude of the OT-I responses was similar, suggesting that not all of the OT-Is transferred at high numbers were used. So, transfer of low numbers of OT-Is was still above a threshold where they could still demonstrate potent inhibition of the host response to other influenza A virus epitopes after infection. Importantly, simultaneous infection with both wild-type HK and HK-OVA viruses did not fully overcome the immunodomination of DbPA224 and KbPB1703 responses by OT-I CTL. This suggests that in this model, naive T cell precursor frequency is a more significant factor contributing to immunodomination rather than the amount of Ag presented. It was interesting to note that a proportion of transferred OT-I were capable of making IFN- upon peptide stimulation despite not receiving Ag-specific stimulation (data not shown). Perhaps a reason for the potency of OT-I competition is they are able to be recruited and acquire effector function very early. It has been suggested that early production of IFN- can provide a competitive advantage for a given CTL population, enabling this population to dominate the T cell response to infection (6). Such early competition is likely to impact on those epitopes where Ag will be more limited. Another possible explanation for the immunodomination is better viral control due to early recruitment of OT-I CTL lowering overall levels of Ag. The antiviral effects of IFN- production by OT-Is may play a role in explaining why after coinfection with HK and HK-OVA viruses, the DbNP366-specific response was restored in the infected lung, yet the DbPA224-specific response was only partially restored. IFN- production by OT-Is could limit replication of both viruses in the lung. As there is more NP than PA produced per virion after infection (21), coinfection with both HK-OVA and HK possibly still results in enough NP366, but not enough PA224 to overcome the suppressive effects of the OT-I CTL.
Overall, the results are in accordance with the idea that altering the size of the precursor pool for a particular T cell set can modify unrelated virus-specific CD8+ T cell immunodominance hierarchies (2, 7, 8, 21). In general, it seems that the factors determining the magnitude of any particular epitope-specific CD8+ T cell response are robust. Although such profiles can be changed by emphasizing or eliminating other Ag-specific CD8+ sets, the magnitude of the effect is likely to be such that the effective development of either T cell memory or a protective recall response will not be substantially compromised.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by an Australian Postgraduate Scholarship awarded to M.R.J.; a National Health and Medical Research Council Burnet Fellowship awarded to P.C.D.; a National Health and Medical Research Council RD Wright Fellowship awarded to S.J.T.; Science Technology, Innovation funds from the Government of Victoria, Australia; and U.S. Public Health Service Grants AI29579 and ALSAC at St. Jude Children’s Research Hospital. 
2 Address correspondence and reprint requests to Dr. Stephen J. Turner, Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia. E-mail address: sjturn{at}unimelb.edu.au
3 Abbreviations used in this paper: Tg, transgenic; BAL, bronchoalveolar lavage; i.n., intranasal; MDCK, Madin-Darby canine kidney; NA, neuraminidase; NP, nucleoprotein; PA, acidic polymerase; PB1, basic polymerase subunit 1.
Received for publication December 14, 2005. Accepted for publication June 16, 2006.
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synthesis increases markedly over the course of infection and correlates with immunodominance. J. Immunol. 173: 456-462. This article has been cited by other articles:
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  L. E. Valentine, J. T. Loffredo, A. T. Bean, E. J. Leon, C. E. MacNair, D. R. Beal, S. M. Piaskowski, Y. C. Klimentidis, S. M. Lank, R. W. Wiseman, et al. Infection with "Escaped" Virus Variants Impairs Control of Simian Immunodeficiency Virus SIVmac239 Replication in Mamu-B*08-Positive Macaques J. Virol., November 15, 2009; 83(22): 11514 - 11527. [Abstract] [Full Text] [PDF]
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 A. Lev, P. Dimberu, S. R. Das, J. C. Maynard, C. V. Nicchitta, J. R. Bennink, and J. W. Yewdell Efficient Cross-Priming of Antiviral CD8+ T Cells by Antigen Donor Cells Is GRP94 Independent J. Immunol., October 1, 2009; 183(7): 4205 - 4210. [Abstract] [Full Text] [PDF]
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B. Moltedo, C. B. Lopez, M. Pazos, M. I. Becker, T. Hermesh, and T. M. Moran Cutting Edge: Stealth Influenza Virus Replication Precedes the Initiation of Adaptive Immunity J. Immunol., September 15, 2009; 183(6): 3569 - 3573. [Abstract] [Full Text] [PDF]
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A. Sexton, R. De Rose, J. C. Reece, S. Alcantara, L. Loh, J. M. Moffat, K. Laurie, A. Hurt, P. C. Doherty, S. J. Turner, et al. Evaluation of Recombinant Influenza Virus-Simian Immunodeficiency Virus Vaccines in Macaques J. Virol., August 1, 2009; 83(15): 7619 - 7628. [Abstract] [Full Text] [PDF]
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 J. D. Mintern, S. Bedoui, G. M. Davey, J. M. Moffat, P. C. Doherty, and S. J. Turner Transience of MHC Class I-restricted antigen presentation after influenza A virus infection PNAS, April 21, 2009; 106(16): 6724 - 6729. [Abstract] [Full Text] [PDF]
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K. M. Grebe, H. D. Hickman, K. R. Irvine, K. Takeda, J. R. Bennink, and J. W. Yewdell Sympathetic nervous system control of anti-influenza CD8+ T cell responses PNAS, March 31, 2009; 106(13): 5300 - 5305. [Abstract] [Full Text] [PDF]
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G. H. Y. Lin, B. J. Sedgmen, T. J. Moraes, L. M. Snell, D. J. Topham, and T. H. Watts Endogenous 4-1BB Ligand Plays a Critical Role in Protection from Influenza-Induced Disease J. Immunol., January 15, 2009; 182(2): 934 - 947. [Abstract] [Full Text] [PDF]
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E. Ingulli, C. Funatake, E. L. Jacovetty, and M. Zanetti Cutting Edge: Antigen Presentation to CD8 T Cells after Influenza A Virus Infection J. Immunol., January 1, 2009; 182(1): 29 - 33. [Abstract] [Full Text] [PDF]
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M. R. Jenkins, J. Mintern, N. L. La Gruta, K. Kedzierska, P. C. Doherty, and S. J. Turner Cell Cycle-Related Acquisition of Cytotoxic Mediators Defines the Progressive Differentiation to Effector Status for Virus-Specific CD8+ T Cells J. Immunol., September 15, 2008; 181(6): 3818 - 3822. [Abstract] [Full Text] [PDF]
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M. F. Kotturi, I. Scott, T. Wolfe, B. Peters, J. Sidney, H. Cheroutre, M. G. von Herrath, M. J. Buchmeier, H. Grey, and A. Sette Naive Precursor Frequencies and MHC Binding Rather Than the Degree of Epitope Diversity Shape CD8+ T Cell Immunodominance J. Immunol., August 1, 2008; 181(3): 2124 - 2133. [Abstract] [Full Text] [PDF]
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P. Castiglioni, D. S. Hall, E. L. Jacovetty, E. Ingulli, and M. Zanetti Protection against Influenza A Virus by Memory CD8 T Cells Requires Reactivation by Bone Marrow-Derived Dendritic Cells J. Immunol., April 1, 2008; 180(7): 4956 - 4964. [Abstract] [Full Text] [PDF]
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 W. Kastenmuller, G. Gasteiger, J. H. Gronau, R. Baier, R. Ljapoci, D. H. Busch, and I. Drexler Cross-competition of CD8+ T cells shapes the immunodominance hierarchy during boost vaccination J. Exp. Med., September 3, 2007; 204(9): 2187 - 2198. [Abstract] [Full Text] [PDF]
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K. Kedzierska, J. Stambas, M. R. Jenkins, R. Keating, S. J. Turner, and P. C. Doherty Location rather than CD62L phenotype is critical in the early establishment of influenza-specific CD8+ T cell memory PNAS, June 5, 2007; 104(23): 9782 - 9787. [Abstract] [Full Text] [PDF]
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), or EL4 cells pulsed with 1 µM OVA257 (
), NP366 (•), or PA224 (
) peptide. Also shown is specific lysis of EL4 cells infected with 10 multiplicity of infection of PR8 (
) or PR8-OVA (
), PB1703 (
) for 5 h in the presence of brefeldin A, then stained with anti-CD8