In the present study it was demonstrated that NP418-specific polyclonal CTL populations recognized this hypervariable epitope with high functional avidity.
Since it was shown that CTL, which recognize their epitopes with high functional avidity are superior in eliminating virus-infected cells or tumor cells (1
), we hypothesized that CTL with high functional avidity could exert higher selective pressure on influenza viruses and therefore drive diversification of CTL epitopes more efficiently than low-functional-avidity CTL, allowing the virus to escape from recognition by specific CTL.
First, we established an assay with which we could determine functional avidity of CTL specific for nine different epitopes in a high-throughput fashion. We wished to use only a limited number of HLA-matched BLCL as stimulator cells for the induction of IFN-γ in the effector cell populations, and therefore, we demonstrated that similar EC50
s were obtained regardless of the BLCL that was used for stimulation. In addition, the values obtained in the IFN-γ ELIspot assay were similar to those obtained in another functional test, the classical 51
Cr release assay. These results are in contrast with previous observations which indicated that the induction of cytolytic activity and IFN-γ production in cytomegalovirus- and HIV-specific CTL do not have the same peptide concentration thresholds (6
). These differences may be explained by the nature of infection (chronic versus acute), the viruses and/or the epitopes that were studied, or the source and nature of the T cells that were used for the analysis (ex vivo T cells versus in vitro-expanded T cells) (7
). We wanted to use polyclonal effector cell populations, which were obtained after stimulation with virus-infected cells, since this would better reflect the in vivo situation than, for example, the use of single T-cell clones specific for the nine peptides that were tested. Indeed, individual CTL clones that recognize a particular epitope could do so with different functional avidities (50
). However, we have tested the EC50
s for some T-cell clone-peptide combinations and these were in the same order of magnitude as those observed with the polyclonal T cell populations (data not shown).
The data obtained with PBMC in vitro may also have implications for peripheral T-cell memory in the airways, since it has been shown that memory T-cell populations in the lung are recruited from the circulation (19
Previously, it has been shown that functional avidity can mature during the course of acute infection (43
). Since, in our study, PBMC were collected outside the epidemic period without any influenza activity, it is unlikely that the study subjects experienced acute infections. Most likely, virus-specific memory CTL were studied that had reached their maximum functional avidity.
The range of EC50
s varied widely with the CTL specific for the NP265
epitope on the high end and those specific for the NP418
epitope on the low end of the spectrum. Seven of the nine epitope-specific CTL populations exhibited a narrow range of functional avidity. This may reflect the oligoclonal nature of these CTL responses, as was demonstrated for the M158
-specific CTL response, which is dominated by T cells carrying the Vβ17+
T-cell receptor (25
We feel that it is conceivable that viruses have an advantage if they can escape from CTL with high functional avidity that require up to 1,000 times less MHC class I peptide complexes for their activation than some of the other CTL. A time window is created in which (mutant) virus replication is unaffected by these efficient CTL, allowing more progeny virus to be produced until CTL with lower functional avidity ultimately eliminate the infected cell. In this scenario, more virus is produced during a longer period of time.
Others have demonstrated with a number of different CTL clones specific for various HIV proteins that their antiviral efficiency is not dependent on their functional avidity per se but on that of the protein that is recognized. In particular, CTL specific for the Nef protein, which is expressed early in the virus replication cycle, were more efficient in inhibiting virus replication (50
). Indeed, the time point after infection at which HIV-infected cells became sensitive to CTL-mediated killing had a profound effect on virus shedding in vitro and in vivo (46
). Although in the latter studies the functional avidity was not studied, they clearly indicated that the kinetics of CTL recognition was determined by the kinetics of protein expression in the infected cells. Of course, the kinetics of lentivirus replication do not apply to influenza virus replication, where all proteins are expressed within 4 h postinfection. Therefore, CTL with high functional avidity may be more important in the control of influenza virus than of HIV.
Thus, escape from high functional avidity CTL seems a plausible scenario from which the virus benefits the most. This reasoning is in agreement with findings in the SIV macaque model (30
). It was found that CTL with high functional avidity eliminated virus-infected cells most efficiently and exerted strong intrahost selection. As a result, mutations were observed in the epitopes recognized by high-functional-avidity CTL, allowing the virus to escape recognition by these CTL. Thus, this chronic infection model showed that there was a strong correlation between T-cell functional avidity and variability in CTL epitopes.
The superior effector function of T cells with high functional avidity have been documented previously, and it most likely is related to multiple mechanisms for the elimination of virus-infected cells (18
), which in turn may contribute to the emergence of CTL escape mutants, as was shown in mice transgenic for the T-cell receptor specific for the influenza A virus NP366-374
Following respiratory infection with simian virus 5 or vaccinia virus, it was found that the CTL response early after infection is of high functional avidity compared to CTL that arise later after infection (24
). In contrast, CTL specific for the Epstein-Barr virus early antigens recognized their epitopes with lower functional avidity and were more immunodominant and more efficient in eliminating infected cells than those specific for late antigens. Probably the efficiency of antigen processing during the lytic stage of the herpes virus infection cycle had affected recognition by CTL specific for the L antigens (37
We now provide evidence that influenza viruses that cause acute respiratory tract infections in a significant portion of the human population are also subject to diversification in CTL epitopes driven by CTL with high functional avidity when immune pressure in the population is sufficiently high. In a population that has been exposed multiple times to the virus and in which a strong CTL− herd immunity has been established, a difference of a couple of hours before infected cells become sensitive to CTL killing could result in a marked advantage to the virus in selected individuals. In a theoretical model, prolonged viral shedding in a small portion of the human population with the corresponding HLA haplotype provided sufficient advantage for rapid fixation of the CTL escape mutants at the population level (21
). Other factors may also contribute to the variability in CTL epitopes. Of special interest is the fact that some of the variable epitopes overlap and are presented by different HLA molecules, like the NP380
epitopes. The NP418
epitope was also found to bind to HLA-B7 in addition to HLA-B*3501 (16
). Despite the evidence for selective pressure in 9-mer amino acid sequences constituting influenza virus CTL epitopes (5
) and for a correlation between CTL functional avidity and variability in CTL epitopes (this study), there are exceptions. For example, the HLA-A*0201-restricted M158-66
epitope is immunodominant, elicits CTL with high functional avidity in a large portion of the human population, and yet, is highly conserved. A mutational analysis of this epitope indicated that this epitope is under functional constraints, which may limit escape from CTL. It is of interest that alanine replacements were tolerated to certain extents at each residue of the 9-mer except for the second, which constitutes the anchor residue of the epitope. More conservative substitutions at this position had less dramatic effects on viral fitness but did not prevent recognition by M158-66
-specific CTL (5
). The amino acid substitution at the anchor residue of the HLA-B*2705-restricted NP383-391
epitope appeared detrimental to viral fitness and was only tolerated in the presence of functionally compensating comutations (38
). These findings indicate that the virus cannot simply evade host immunity at the cost of viral fitness. In this respect, it is of interest that the otherwise hypervariable NP418
epitope retained its anchor residues, which could not be replaced without the loss of viral fitness (5
The functional avidity of CTL specific for conserved influenza virus epitopes also correlated with the magnitude of the CTL responses in vitro. This finding is in concordance with previous studies that showed a correlation between the functional avidity and the immunodominance of the CTL response specific for minor histocompatibility antigens in a mouse model (53
It is possible that the study subjects had been exposed less frequently to mutant CTL epitopes than conserved epitopes, which may have affected the magnitude of the CTL responses in vitro. This might explain the loss of correlation between functional avidity and immunodominance when the variable epitopes are included in the comparison (Fig. ).
Studies on influenza A virus-specific CTL responses in mice also identified a role for functional avidity in establishing immunodominance hierarchies among various epitope-specific T cells (3
). However, it is known that the hierarchy of primary and secondary CTL response can differ and other factors, like epitope abundance, differential antigen presentation, or limitations in the T-cell response must be involved (3
It is likely that immunodominant epitopes also bind to their corresponding MHC class I molecules with high affinity, which in turn could result in preferential peptide loading in the endoplasmic reticulum and sustained presentation to specific CTL. Mathematical models also suggested that small differences in the initial time of activation have a large impact on the magnitude of the response (52
We hypothesize that in the battle between influenza virus and its host, the virus evades recognition by CTL with high functional avidity by accumulating amino acid substitutions in CTL epitopes, allowing the virus to replicate to higher titers and for prolonged periods of time in selected individuals. The diversification in epitopes recognized by CTL with high functional avidity might be of importance for the successful persistence of influenza A viruses in the human population.