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Hidden Epitopes Emerge in Secondary Influenza Virus-Specific CD8⁺ T Cell Reponses¹

Paul G. Thomas^*, Scott A. Brown^*, Rachael Keating^*, Wen Yue^*, Melissa Y. Morris^*, Jenny So^*, Richard J. Webby and Peter C. Doherty^2,*

^* Department of Immunology and Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, TN 38105

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Influenza A virus-specific CD8⁺ T cell responses in H2^b mice are characterized by reproducible hierarchies. Compensation by the D^bPB1-F2₆₂ epitope is apparent following infection with a variant H3N2 virus engineered to disrupt the prominent D^bNP₃₆₆ and D^bPA₂₂₄ epitopes (a double knockout or DKO). Analysis with a "triple" knockout (TKO) virus, which also compromises D^bPB1-F2₆₂, did not reveal further compensation to the known residual, minor, and predicted epitopes. However, infection with this deletion mutant apparently switched protective immunity to an alternative Ab-mediated pathway. As expected, TKO virus clearance was significantly delayed in Ab-deficient MHC class II^–/– and Ig^–/– mice, which were much more susceptible following primary, intranasal infection with the TKO, but not DKO, virus. CD8⁺ T cell compensation was detected in DKO, but not TKO, infection of Ig-deficient mice, suggestive of cooperation among CD8⁺ T cell responses. However, after priming with a TKO H1N1 mutant, MHC II^–/– mice survived secondary intranasal exposure to the comparable H3N2 TKO virus. Such prime/challenge experiments with the DKO and TKO viruses allowed the emergence of two previously unknown epitopes. The contrast between the absence of compensatory effect following primary exposure and the substantial clonal expansion after secondary challenge suggests that the key factor limiting the visibility of these "hidden" epitopes may be very low naive T cell precursor frequencies. Overall, these findings suggest that vaccine approaches using virus vectors to deliver an Ag may be optimized by disrupting key peptides in the normal CD8⁺ T cell response associated with common HLA types.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Virus-specific CD8⁺ T cell responses are comprised of distinct epitope-specific sets that expand to varying degrees following Ag challenge, leading to numerically defined immunodominance hierarchies (1). The factors that determine such hierarchies are still poorly understood, though a variety of explanations have been advanced (1, 2, 3, 4, 5, 6, 7). From a practical point of view, though, establishing how both quantity and quality impact on CTL effector function is clearly important as we seek to design T cell vaccines that promote early virus clearance (2) or are useful for cancer immunotherapy. In particular, assessing the nature of immunodominance in compensatory CD8⁺ T cell responses where one epitope is mutated or eliminated has special significance for viruses that can vary rapidly, such as influenza and HIV, and for tumor immunity. Several factors have been considered to play a role in immunodominance, including: the character of TCRs and TCR repertoire availability, Ag density, the efficiency of Ag processing and presentation, peptide/MHC "off rates," and "immunodomination" effects by prominent epitopes, including competition for both CD8⁺ T cell access to APCs and for secreted factors that promote the expansion of other Ag-specific sets (1, 3, 4, 5).

An impressive but unusual immunodominance hierarchy has been identified in C57BL/6 (B6, H2^b) mice for the influenza nucleoprotein (NP)³ peptide presented by H2D^b (D^bNP₃₆₆). Although the D^bNP₃₆₆ epitope induces a response approximately equivalent in magnitude to that associated with the viral acid polymerase (PA) D^bPA₂₂₄ following primary infection, the D^bNP₃₆₆-specific population expands to an 10x higher level following secondary challenge. One set of experiments showed that while both epitopes are expressed on the dendritic cells (DCs) thought to be the sole APCs mediating primary CD8⁺ T cell stimulation, only D^bNP₃₆₆ can be found on other cell types, such as macrophages or the virus-infected epithelium, that can (at least in vitro) be shown to target effector or memory CD8⁺ T cells (6). However, a further study using influenza A viruses that had been engineered to express equivalent amounts of NP_366–374 and PA_224–236 in the viral neuraminidase protein, while at the same time disrupting these peptides in their native configurations, suggested that TCR repertoire size and Ag load are key determinants of magnitude for both primary and secondary CD8⁺ T cell responses (7).

The use of such reverse genetics strategies to mutate key peptides in the influenza A virus/B6 mouse model has further potential for illuminating the characteristics of virus-specific immunodominance hierarchies (8, 9). In all, six peptide plus MHC class I glycoprotein (pMHC1) complexes (including D^bNP₃₆₆ and D^bPA₂₂₄) have been identified for H2^b mice infected with the A/PR/8/34 (PR8, H1N1) and A/HKx31 (x31, H3N2) influenza A viruses. In various experiments, viruses that have been engineered to delete the D^bNP₃₆₆- and D^bPA₂₂₄-specific responses have been shown to induce a measure of compensatory expansion by CD8⁺ T cells specific for other epitopes (9). Some have suggested that such compensatory responses are evidence of immunodomination. Additionally, binding algorithms have been used to predict a number of potentially immunogenic influenza virus pMHC1 combinations, some of which have been manipulated to give minimal Ag-specific responses (10).

Robust compensation was observed in the murine lymphocytic choriomeningitis virus model where the removal of a major epitope induced the expansion of subdominant specificities, supporting the view that there is some form of regulatory control exerted by the dominant CD8⁺ set (10, 11). The secondary influenza-specific response in mice primed and boosted with double "knockout" (DKO) H1N1 and H3N2 viruses engineered to disrupt D^bNP₃₆₆ and D^bPA₂₂₄ showed evidence (8) of partial compensation by CD8⁺ T cells specific for the D^bPB1-F2₆₂ epitope that is derived from translation of an alternative reading frame (F2) of the viral polymerase PB1 protein (PB1-F2). This line of investigation has now been extended by the generation of triple knockout (TKO) viruses that also lack the capacity to form the D^bPB1-F2₆₂ peptide-MHC I complex (pMHCI). Particularly in Ig compromised mice the present analysis has revealed strong secondary responses to novel, unpredicted epitopes. Furthermore, the experiments suggest that there may be a measure of CD8⁺/CD8⁺ T cell "help" under some conditions of virus challenge.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Mice, viruses, and infection

Female B6 mice were purchased from The Jackson Laboratory and CD4⁺ T cell-deficient MHC class II^–/– mice and B cell-deficient µMT mice were bred at St. Jude Children’s Research Hospital. All were held under specific-pathogen-free conditions and were first infected between 8 to 10 wk of age. The generation and preparation of wild-type (WT) PR8 and x31 and mutant (PR8(–NP –PA) (PR8-DKO), PR8(–NP –PA –F2) (PR8-TKO), x31(–NP), x31(–PA), x31(–NP –PA) (DKO), x31(–NP –PA –F2) (TKO)), and x31(–NP –PA –F2 –PB1 –NS2) (quintuple knockout or QKO)) recombinant viruses by using the eight-plasmid reverse genetics system has been described previously (9, 11) for the mutation of the D^bNP_366–374 and D^bPA_224–233 epitopes. In the TKO viruses, the WT D^bPB1-F2_62–70 epitope (LSLRNPILV) (12) was altered to LSLRQPILV, an N5Q mutation similar to the ones used for the –NP and –PA constructs. In the QKO virus the WT K^bPB1_703–711 epitope (SSYRRVPGI) was altered to SSFRRVPGI, while the WT K^bNS2_114–121 epitope (RTFSFQLI) was altered to RTFSAQLI. An RMAS cell assay demonstrated that this mutation preventing binding of the epitope to the class I molecule (data not shown). Primed (PR8 variants injected i.p. at 10⁸ 50% egg infectious doses (EID₅₀)) and naive mice were first anesthetized by i.p. injection of 2,2,2-tribromoethanol (Avertin) and then infected i.n. with the indicated doses (usually 10⁶ EID₅₀) of the x31 viruses. The mice were anesthetized again at the time of sampling and then exsanguinated by section of the axillary artery. Inflammatory cell populations were recovered from the infected respiratory tract by bronchoalveolar lavage (BAL) followed by removal of the spleen and lungs.

Ag-specific ELISA

Briefly, microtiter plates (Corning) were coated overnight at 4°C with purified whole influenza HKx31, A/Aichi/68 (H3N2) virus in PBS (8). Influenza-specific IgG1 was detected with a goat anti-mouse IgG alkaline-phosphatase conjugate (Southern Biotechnology Associates) diluted 1/1,000 in PBS with 1% BSA. A substrate (p-nitrophenyl phosphate; Sigma-Aldrich) was added, plates were incubated for 60 min at room temperature for color development, and OD values were determined at 405 nm in an ELISA reader (Molecular Devices). Titers were presented as the highest dilution that yielded an OD three times higher than that for a 1/100 dilution of preimmune serum.

Flow cytometry

Virus-specific CD8⁺ T cell responses were analyzed by flow cytometry. For peptide stimulation-cytokine (PepC) assays, lymphocytes were first incubated with the indicated peptide at 1 µM for 5 h in the presence of 10 µg/ml brefeldin A, fixed with formaldehyde, and stained with CD8 allophycocyanin (clone 53-6.7; BD Pharmingen), IFN- PE (BD Pharmingen catalog no. 554412), and TNF FITC (BD Pharmingen catalog no. 554418). The established influenza epitopes tested were D^bNP_366–374, D^bPA_224–233, D^bPB1-F2_62–70, K^bPB1_703–711, and K^bNS2_114–121. The putative epitopes examined were P1, K^bPB1_214–221, P2, K^bPB2_358–365, P3, K^bPA_238–245, P4, K^bPA_300–307, and P5, K^bPB2_689–696. Autofluorescence was gated out using the FL3 channel. The data were acquired on a BD Biosciences FACSCalibur and analyzed using CellQuest software.

RMAS cell assay

RMAS cells were incubated for 16 h at 31°C followed by incubation with the tested peptides for 30 min at room temperature (concentrations ranging from 100 µM to 4 nM). The cells were then placed at 37°C for 3 h and stained with fluorescently labeled anti-H-2K^b (AF6.88.5.3) or anti-H-2D^b (28.14.85) Abs and analyzed on a flow cytometer.

Viral titer determination

Lung homogenates were titered by plaque assay on Madin-Darby canine kidney (MDCK) cells. Near confluent 25-cm² monolayers of MDCK cells were infected with 1 ml of homogenate or dilution of homogenate (in general, six dilutions of lungs were tested) for 1 h at 37°C. Cells were washed with PBS, 3 ml of MEM containing 1 mg/ml L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Worthington Biochemical) and 0.9% agarose was added, and cultures were incubated at 37°C with 5% CO₂ for 72 h. Plaques were visualized with crystal violet.

ELISPOT analysis

The ELISPOT protocol was used to estimate the total size of IFN--producing CD8⁺ T cell populations in the spleen following stimulation with virus-infected APCs as established in Ref. 8 . Purified CD8⁺ T cells were exposed to APCs infected with the indicated viruses and the number of IFN- producers was measured as spots per 10⁶ input cells after 48 h at 37°C.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
These experiments use prime/boost studies with WT and genetically manipulated variants of the serologically distinct PR8 (H1N1) and HKx31 (H3N2) influenza A viruses. The DKO viruses were mutated to preclude presentation of the D^bNP₃₆₆ and D^bPA₂₂₄ epitopes and the TKO viruses were additionally compromised for D^bPB1-F2₆₂, whereas a QKO virus also disrupted K^bPB1₇₀₃ and K^bNS2₁₁₄. The HKx31 deletion mutants were used for respiratory challenge of naive (primary) or immune (secondary) B6 mice that had been infected i.p. with the homologous, serologically noncross-reactive PR8 variant. The interval between i.p. priming and i.n. challenge was at least 6 wk.

Compensation analysis for predicted peptides

Prior intracellular cytokine staining (ICS) analysis of primary CD8⁺ T cell response profiles of DKO virus-infected B6 mice failed to show any significant compensatory effect resulting from the disruption of D^bNP₃₆₆ and D^bPA₂₂₄, though the analysis was confined to the previously characterized pMHCI complexes (8). These experiments were repeated and extended to look for responses to novel epitopes identified (11) by using a peptide algorithm. Primary exposure to either the DKO (Fig. 1A) or the TKO virus (Fig. 1B) gave no indication that any of these predicted epitopes (P1–P5; see Materials and Methods) were associated with the generation of significant CD8⁺ T cell responses. In addition, the responses to the known, "minor" K^bNS2₁₁₄ and K^bPB1₇₀₃ epitopes did not increase in magnitude following secondary challenge, though we did see the increased D^bPB1-F2₆₂-specific CD8⁺ T cell numbers found previously (8) for the DKO prime/boost (Fig. 1C). Surprisingly, the priming and challenge of mice with the TKO viruses was not associated with an enhanced recall response to any predicted (11) or previously characterized minor epitope (Fig. 1D).

View larger version (39K): [in this window] [in a new window]   FIGURE 1. Analysis of compensatory responses to DKO and TKO viruses using the ICS (A–F) and ELISPOT (G–I) assays. A–D, Analysis of epitope-specific responses in spleen for the day 10 primary (A and B) and day 8 secondary (C and D) responses to the DKO (A and C) or TKO (B and D) viruses. The predicted epitopes (10 ) tested were KbPB1214–221 (P1), KbPB2358–365 (P2), KbPA238–245 (P3), KbPA300–307 (P4), and KbPB2689–696 (P5). E and F, Pooled (E) and individual (F) BAL samples were also tested following primary (E) or secondary (F) challenge with the TKO viruses. G–I, ELISPOT analysis for total IFN- -secreting CD8+ T cells. Spleens from virus infected mice were removed following day 10 primary DKO (G), day 10 primary TKO (H), or day 8 secondary TKO (I) challenge and stimulated with splenocytes infected with WT, DKO, or TKO viruses. All data are representative of at least two independent experiments.*, p < 0.05.

We observed that spleen cells from DKO-infected mice stimulated with cells infected with WT virus showed almost equivalent levels of response to CD8⁺ T cells from mice infected with WT virus (Fig. 1G, bars labeled WT stim). The possibility that infection with the DKO virus might allow the better presentation of "minor" epitopes, leading in turn to some compensatory response, was also suggested by the observation that a larger response was seen following stimulation of DKO-primed T cells with DKO (vs WT)-infected APCs (Fig. 1G). Neither of these compensation effects was seen following ELISPOT analysis of spleen cells recovered at the peak of the primary (Fig. 1H) response to the TKO virus. However, there was a partial compensatory effect observed in the secondary response to the TKO virus (Fig. 1I), though it was not as extensive as the compensation observed for the secondary response to the DKO virus (8). In our hands, the ELISPOT assay is less sensitive and always gives much lower numbers (generally 3x less) than the cumulated total for ICS, where the cell counts also approximate those derived from tetramer staining (Ref. 8 and P. G. Thomas and S. A. Brown, unpublished data).

This analysis of DKO and TKO spleen CD8⁺ T cell response profiles by ICS (Fig. 1, A–C) thus failed to show any compensatory role for epitopes that do not normally emerge following infection with WT influenza A viruses (Fig. 1, A–D). We also analyzed the BAL because, under some conditions of defective response (MHC II^–/– mice), a smaller number of residual CD8⁺ T cell responders can sometimes be enriched in the virus-infected lung (13). Again, analyzing the BAL populations recovered from B6 mice sampled after primary (Fig. 1E) or secondary (Fig. 1F) challenge with the TKO virus did not show any evidence of compensatory responses to either "predicted" or well-characterized epitopes.

Relative pathogenicity of knockout viruses

Primary i.n. infection of B6 mice with the HKx31 TKO variant caused less severe weight loss than either the WT or the DKO virus (Fig. 2A). This could be thought to reflect that the PB1-F2 mutation may have diminished the replicative capacity of the TKO virus, though no such effect was apparent in MDCK cultures or embryonated hen’s egg (data not shown) and there is no obvious reason why a conservative single residue substitution in the PB1-F2 alternative reading frame should in any way disrupt the function of the influenza virus polymerase complex. The function of the PB1-F2 gene product in influenza infection is still poorly understood, so it is possible that this mutation may be mediating an immunological effect via modulation of PB1-F2 activity. However, the lung titers from B6 mice infected with the WT, DKO, and TKO viruses were not significantly different on days 3 and 5, suggesting no defect in the growth of the KO viruses as a result of their mutations, though the TKO variant was cleared more rapidly coincident with the emergence of the adaptive response (B6 mice; Table I).

View larger version (13K): [in this window] [in a new window]   FIGURE 2. Weight loss in the primary response to the HKx31, DKO, or TKO virus in WT B6 (A), Ig–/– µMT (B), and MHC II–/– (C) mice. The results are representative of at least three independent experiments.

View larger version (9K): [in this window] [in a new window]   FIGURE 3. Minor increase in influenza specific IgG1 in the primary response to TKO virus on day 7 (A) but not day 10 (B). The ELISA results are representative of two independent experiments. *, p < 0.05.

The lack of compensation observed in TKO virus-infected mice might have been due to the more rapid clearance and increased Ig responses observed (Table I, B6 mice, and Fig. 3). Thus, Ig-deficient MHC II^–/– (14) and Ig^–/– µMT mice were also used to test the possibility that a more prolonged and severe infectious process (Fig. 2 and Table I) promotes enhanced compensatory CD8⁺ T cell responses to minor epitopes. Unlike the B6 profile (Fig. 1A), primary infection of both the MHC II^–/– and µMT mice induced a compensatory D^bPB1-F2₆₂ response following challenge with the DKO virus (Fig. 4, A and B). This was also seen for K^bNS2₁₁₄ in the MHC II^–/– mice, but not the Ig^–/– µMT mice, given the DKO virus (Fig. 4B). The latter effect, which may also be seen in some experimental situations for the K^bPB1₇₀₃ epitope, was not found at all for the TKO virus (Fig. 4, A and B). Again (Fig. 1), there was no evidence that the "predicted" pMHCI complexes (11) were being recognized following infection with either the DKO or the TKO virus (data not shown).

View larger version (15K): [in this window] [in a new window]   FIGURE 4. Compensation in immunodeficient mice following primary DKO, but not TKO, virus infection as well as in both DKO and TKO secondary challenge. BAL from Ig–/– µMT mice on day 10 (A), spleens from MHC II–/– mice on day 10 after infection (B), and spleens from MHC II–/– mice on day 8 after secondary infection (C) screened with specific peptides by intracellular cytokine staining for IFN-. The results are representative of at least three independent experiments. *, p < 0.05.

Identification of new epitopes

The results so far still provided no explanation for the apparent compensatory effect seen in the "whole virus" ELISPOT analysis (Fig. 1, G–I). Recent analysis (15) using an overlapping peptide scan indicated that four regions of viral proteins not associated with known epitopes could potentially stimulate T cells from infected mice (NP_211–225, PB2_196–210, PB2_91–105, and HA_441–445). Analysis of primary infection showed some response to all four of the peptides, with NP_211–225 and PB2_196–210 inducing the strongest responses in mice infected with the WT, DKO, and TKO viruses (see Fig. 6A and data not shown). Additional experiments using the RMAS assay (Fig. 5) identified K^bNP_217–225 as the NP_211–225 associated epitope, but the minimal epitope within PB2_196–210 was difficult to identify. This region contains PB2_198–206 (line marked "C." in Fig. 5A), the sequence previously identified as a mimotope for the K^bPB1₇₀₃ epitope (16). We were unable to show activity associated with PB2_198–206 but did see partial activity (when compared with the larger peptide) from PB2_197–206 and PB2_196–207 (lines marked "E." and "H.," respectively, in Fig. 5A). As demonstrated previously, PB2_198–206 bound both K^b and D^b. We found a similar "dual" binding profile for PB2_197–206, but PB2_196–207 displayed no affinity for K^b and only bound D^b at very high concentrations. Because it appears that there may be two distinct epitopes in this region, we used the larger peptide for our assays. Studies are ongoing to try and resolve the specific restriction elements in this region.

View larger version (26K): [in this window] [in a new window]   FIGURE 6. Compensation by novel minor epitopes for secondary responses in B6 and MHC II–/– mice. Note that the values on the y-axes vary for the different panels. Primary (day 10) spleen (A and C) and BAL (B and D) and secondary (day 10) spleen (E and G) and BAL (F and H) ICS responses in B6 (A, B, E, and F) and MHC II–/– (C, D, G, and H). The epitopes/peptides tested were KbNP217–225, PB2196–210, PB291–105, and HA441–455. The data are representative of at least two independent experiments and significant differences (*, p < 0.05) are related to values for the WT virus-infected mice.

View larger version (13K): [in this window] [in a new window]   FIGURE 5. A, Mapping the response down within the NP210–225 and PB2196–210 peptide sequences. The results are presented as the percentage of the ICS response to the original 15-mer peptides (100%). B, RMAS cell assay of the peptides from A. C, Comparison of the day 10 ICS response profiles in the B6 spleen following primary QKO or HKx31 virus infection. Note the lack of variance in activity of the PB2196–210 peptide in the QKO vs the WT virus-infected mice.

Robust compensation in secondary responses

The recall responses to K^bNP₂₁₇ and PB2₁₉₆ induced by the HK-DKO and HK-TKO viruses in B6 mice primed with the homologous PR8 variants were significantly higher than those associated with the WT infection (Fig. 6, E and F). These enhanced minor responses did not fully compensate for the loss of the dominant D^bNP₃₆₆, D^bPA₂₂₄, and D^bF2₆₂ populations but were individually comparable in magnitude to the WT K^bPB1₇₀₃ response (compare with Fig. 1). By contrast, the MHC II^–/– mice showed increased counts (relative to the B6 controls; Fig. 6, A and B) for both the K^bNP₂₁₇- and PB2₁₉₆-specific sets following primary WT, DKO, and TKO infection, but there were no differences between the three viruses in this regard (Fig. 6, C and D). After secondary DKO and TKO virus challenge, however, there was robust compensation (relative to the WT virus) by both the K^bNP₂₁₇ and PB2₁₉₆ epitopes (Fig. 6, G and H). These secondary infection data, combined with the primary infection data from Fig. 4, suggest that delayed viral clearance in MHC II^–/– mice correlates with increased levels of compensation to minor epitopes in both primary and secondary responses. The prominence of this effect in secondary responses likely results from the increased precursor frequency of the epitope-specific populations.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Infection with viruses disrupted for two (DKO) or three (TKO) prominent pMHC l epitopes suggests that Ag persistence and a certain minimal response quality must be generated to allow the compensatory expansion of subdominant epitope-specific populations. These data also support earlier indications that there is a complex interplay between CD8⁺ T cell-mediated and humoral immunity in the control of influenza A virus infections (17). Absence of the D^bNP₃₆₆, D^bPA₂₂₄, and D^bPB1-F2₆₂ epitopes seemed to generate a predominantly Ab-based primary response, with the result that the virus is cleared more rapidly from immunologically competent Ig^+/+ mice. The fact that this in turn leads to a relative loss of residual "minor" CD8⁺ T cell responses in conventional mice infected with the TKO viruses could be thought to reflect a lower Ag load; but when the experiment was repeated in Ab-deficient mice that cleared this virus more slowly, we still failed to see any evidence of the compensation found previously with the DKO virus. The idea that the overall lack of a substantial compensatory CD8⁺ T cell response in TKO-infected mice was a result of Ab dominance is thus not tenable; the loss of these three key epitopes effectively abrogated protective cell-mediated immunity following primary infection. By contrast, the secondary response to the TKO virus, where the precursor frequencies before virus challenge will be much higher than in the naive situation, induces a much more robust compensatory CD8⁺ T cell response in both B6 and MHC II^–/– mice.

These experiments also provide the somewhat surprising insight that there could be interactive "helper" effects between the various CD8⁺ T cell populations. The absence of certain "strong" responders seems to diminish the expansion of "weaker" CD8⁺ sets. This is likely to reflect the specific quality of one or more of the deleted responses rather than a direct quantitative effect. One possibility is that cytokines like IFN- (4) or IL-2 (18) produced by "strong" responders function to promote "activated" microenvironments that enhance the overall antigenicity of the DC stimulators in lymphoid tissue. Such a mechanism would normally, of course, be thought to operate through CD4⁺ help. However, neither the DKO nor the TKO virus is modified in a way that is likely to compromise the generation of MHC II-restricted epitopes (15), and the observed compensation in the secondary response by MHC II^–/– mice is obviously CD4⁺ T cell independent (somewhat in contrast to other model systems where secondary CD8⁺ T cell responses are extremely dependent on priming in the presence of CD4⁺ T cells (19)). Perhaps a minimum overall level of CD8⁺ T cell expansion is required to sustain a sufficient response to any epitope past the initial phase of infection. Although early inflammation and "danger" signals no doubt provide the initial milieu for APC induction and, transitively, CD8⁺ T cell activation, the longer-term maintenance of "stimulatory microenvironments" in the lymph node may require a more prolonged "cytokine/chemokine swarm."

At this stage of understanding, is it possible to develop some useful synthesis of what determines a CD8⁺ T cell immunodominance hierarchy? Any explanatory model of immunodominance must take into account influences due to: 1) the concentration of the presented epitope; 2) the size of the available CD8⁺ T cell repertoire; 3) the division program activated after the initial priming; 4) competition and cooperation between different epitope-specific responses; and 5) compensatory effects in some, but not other, experimental situations.

Attempting to account for observations made by a number of investigators, Antia et al. (20, 21) proposed a mathematical model for the expansion of epitope-specific CD8⁺ T cells that emphasizes a "program" of Ag-independent division. According to this idea, the Ag-dependent phase primarily involves cell recruitment with immunodominance being a direct reflection of precursor compartment size and the magnitude of recruitment. Thus, a main determinant of immunodominance in this model is the inherent T cell repertoire, an idea that is supported by our own experiments with "epitope-shifted" (to the viral neuraminidase) influenza A viruses (7), though it does not take into account the central role for Ag density that was also suggested by these studies.

Other experiments have indicated that primary CD8⁺ T cell stimulation in mice infected with influenza A viruses is mediated via Ag-presenting DCs that move from the virus-infected lung to the regional lymph nodes during the first 36 h after Ag challenge (22). In accord with this observation, Schoenberger and coworkers (23) established an in vitro time threshold of 20 h for naive T cell/DC-pMHCI interaction to induce the full division program (>8 divisions). Kaech et al. (24) came to similar conclusions and showed that shorter DC-T cell contact to give a maximum of approximately three divisions led to an "aborted" differentiation program. Combining these ideas, both Ag-load and the extent of cycling during the window of Ag-dependent proliferation can be thought to influence the immunodominance profile.

Models that emphasize only the events occurring during the first 20–36 h after the initial encounter with a virus can be used to explain the absence of epitope-driven compensatory effects in some experimental situations, though they do not account for other instances where compensation occurs. One consideration with viruses is that the kinetics of antigenicity will vary for the different epitopes. In the influenza model, the "late" Ags are likely to be derived from proteins that are essential for replication but not incorporated at significant concentrations in the virus particle, namely D^bPB1-F2₆₂ and K^bNS2₁₁₄. The input inoculum would thus fail to induce a "very early" response to these epitopes, as they will only be generated following infection and the synthesis of new protein. If the Ag-dependent "window" is lengthened by increased inflammation or increased Ag load, the expectation would be that the most dramatic compensation would be associated with peptides from these "late" proteins. In the case of the DKO viruses, two epitopes that compensated to significant levels in the immunodeficient animals and in the secondary response were D^bPB1-F2₆₂ and K^bNS2₁₁₄ (8), derived from two influenza proteins that are not in the virus particle or are only present at very low levels. However, both the newly characterized NP_217–225 and PB2₁₉₆ peptides would be expected to be present at much higher levels than either PB1-F2₆₂ or NS2₁₁₄ in free virions. The lack of CD8⁺ T cell compensation in Ig-deficient mice following primary virus challenge may thus reflect that the compensatory responses to these epitopes is limited by the relatively small naive precursor frequency, a factor that is less important in secondary responses where Ag density may be the key factor.

It is clearly important that we evolve a more sophisticated understanding of the rules underlying CD8⁺ T cell immunodominance hierarchies. The present results indicate that, at least for the nonpersistent influenza A virus model, a limit is quickly reached in the efficiency of the compensatory response. Although compensation to multiple epitopes was measurable following secondary challenge with the TKO virus, these responses did not compensate numerically for the loss of the WT virus responses and led to increased morbidity and mortality following primary infection of immunodeficient animals. This suggests that, at least for this particular virus with its relatively small genome, the overall available T cell repertoire is limited in extent, a consequence that would have greater implications for persistent pathogens that (like HIV) use a high rate of mutation as an immune escape mechanism. The present deficiency mutants mimic these natural situations and are clearly useful substrates for exploring the consequence of various "knock-in" strategies (7). We may, in fact, be approaching a situation where the measurement of a few parameters that are readily determinable in vitro could allow quantitative prediction of burst size and immunodominance hierarchies for the complex of Ag-specific CD8⁺ T cell responses that are likely to result from the exposure to any infectious agent or potentially immunogenic cancer cell. This will hopefully be of value as we seek to develop better vaccines and immunotherapy protocols based on the exploitation of cell-mediated immunity in particular, where we are trying to develop CD8⁺ effectors specific for "weak" cancer Ags expressed in viral vectors. Diminishing the response to the native virus epitopes may help ensure that the engineered neo-Ags achieve sufficient prominence.

	Acknowledgments

 
We thank Nicole La Gruta and Steven Turner for thoughtful discussion of experiments and the manuscript.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This study was supported by National Institutes of Health Grants AI70251 (to P.D.), AI95357 (to R.W.), and AI065097 (to P.T.), a Burnet Award (to P.D.), and by private donations to the American Lebanese Syrian Associated Charities at St. Jude Children’s Research Hospital.

² Address correspondence and reprint requests to Dr. Peter C. Doherty, Department of Immunology, St. Jude Children’s Research Hospital, 332 N. Lauderdale Street, Memphis, TN, 38105. E-mail address: Peter.Doherty{at}stjude.org

³ Abbreviations used in this paper: NP, nucleoprotein; BAL, bronchoalveolar lavage; DC, dendritic cell; DKO, double knockout; F2, alternative reading frame of PB1 protein; ICS, intracellular cytokine staining; i.n., intranasal(ly); MDCK, Madin-Darby canine kidney; PA, viral acid polymerase; pMHCI, peptide-MHC I complex; QKO, quintuple knockout; TKO, triple knockout virus; WT, wild type.

Received for publication November 17, 2006. Accepted for publication December 21, 2006.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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