In this study, cellular immune responses to a live influenza A virus strain were evaluated in children aged 6 months to 9 years and adults aged 22 to 48 years before and after immunization with TIV or LAIV. After one dose of LAIV, the average percentage of influenza A virus-reactive IFN-γ+ cells in T-cell and NK cell subsets, detected after stimulation with live influenza A virus/Wyoming (H3N2), increased significantly in children aged 5 to 9 years but not in adults. In children aged 5 to 9 years, LAIV was more likely to induce an increase in the percentage of influenza A virus-specific IFN-γ+ CD4 and CD8 T cells than TIV. Age also significantly influenced immune responses to LAIV and TIV. The change (n-fold) in IFN-γ+ T cells induced by LAIV was smaller among the adults than among 5- to 9-year-old children. Significant inverse correlations between the postvaccination change (n-fold) in influenza A virus-reactive IFN-γ cells in various lymphocyte subsets and their prevaccination levels were observed in all age and vaccine groups, except for children given TIV. The strongest correlation was observed in adults given LAIV. Among adults, the baseline levels of IFN-γ+ cells in the CD56dim NK cell subset were associated most directly with change (n-fold) in IFN-γ+ cells after either LAIV or TIV vaccination. Although differences in the pattern of the inverse correlation were observed, the vaccine-induced change (n-fold) in influenza A virus-reactive IFN-γ+ cells in all four lymphocyte subsets, including CD4 and CD8 T cells and CD56bright and CD56dim NK cells, correlated positively with each other in all age and vaccine groups (data not shown). Notably, the cellular immune responses to TIV and LAIV showed no correlation with the day 0 to day 10 change (n-fold) in neutralizing antibody titers against influenza A virus/Wyoming (unpublished data).
In a limited number of influenza vaccine-naïve children younger than age 5, the average percentage of influenza A virus-reactive IFN-γ+ cells increased significantly in the CD8 T-cell and CD56bright NK cell subsets after one dose of TIV, while significant changes were not observed in any T- or NK cell subsets in older children or adults after TIV vaccination. The reason for the different responses in different age groups remains unclear but may be related to the lack of preexisting influenza virus immunity in the youngest age group. Interestingly, the percentages of IFN-γ+ cells detected in the subjects tested at day 10 after a second dose of TIV given 28 days after the first dose were not greater than the levels observed in the subjects tested at day 10 after the first dose. Possible explanations for this unexpected result include a difference in the kinetics of the secondary responses or in the functional status of immune cells that are reexposed to the HA and NA antigens after a recent primary exposure. These issues should be addressed in future studies with larger sample sizes in order to optimize the use of TIV in this youngest age group. However, it is interesting that a similar lack of boosting effect was observed in the same subjects in their antibody-secreting-cell responses to the second dose of TIV (unpublished data).
Presentation of exogenous protein antigen to CD8 T cells, such as the influenza virus proteins in the TIV formulation, requires cross-presentation of the antigen through the major histocompatibility complex class I pathway (44
). Dendritic cells (DCs) are the major antigen-presenting cells that have the capability to cross-present exogenous protein antigens to CD8 T cells (1
). The detection of influenza A virus-specific CD8 T cells following immunization with TIV in children younger than 5 years old suggests that the cross-presentation pathway is functional in this age group. It is unclear why TIV failed to induce a significant T- or NK cell response in older children, although one might speculate that the preexisting influenza virus immunity diminished the ability of DCs to present viral antigen efficiently. Interestingly, LAIV appears to be more likely to induce influenza A virus-specific IFN-γ+
T-cell responses than does TIV, as demonstrated in the 5- to 9-year-old vaccinees. This seems likely to be due to the provision of additional antigens in the form of endogenously synthesized viral proteins in LAIV-immunized subjects. It is also possible that the replication of LAIV in the respiratory tract provides a more favorable environment for DCs and other antigen-presenting cells to present viral antigens to T cells. These findings may have implications for the relative protective effects of LAIV and TIV in children. It will be important to directly compare responses to TIV and LAIV in children younger than 5 years old to determine whether these two vaccines also differ in the capacity to induce T-cell responses in the youngest age group and to examine the efficiency of cross-priming versus conventional endogenous priming for the stimulation of primary major histocompatibility complex class I-restricted responses in influenza-naïve children. It will also be of interest to investigate the shedding of replicating vaccine virus in LAIV recipients and to investigate if vaccine replication is associated with T-cell responses in recipients.
The innate immune response, which is rapid but not considered antigen specific, provides a first line of defense against viral infection and influences the subsequent adaptive T-cell responses (36
). It was reported that in human volunteers experimentally challenged with wild-type influenza virus, infection caused a significant increase in NK cell activity (13
). Despite the importance of innate immunity in host defense against viral infection, most previous immunological studies of influenza vaccines have focused on adaptive immunity, i.e., B-cell and T-cell responses, and rarely on innate immunity. In limited studies of the NK cell response to inactivated influenza vaccine, an enhanced NK cell cytotoxicity has been related to vaccination in some studies (38
), but not others (29
IFN-γ is known for its direct antiviral activity, as well as its immune-regulatory activity (14
). Rapid production of IFN-γ and other inflammatory cytokines by NK cells, including the CD56bright
NK cell subsets, is an important component of the innate immune response (6
). In the current study, we demonstrated that the IFN-γ response of NK cells to influenza A virus antigens can be enhanced by vaccination, especially with LAIV, in the majority of children, as well as in some adults. Based on our previous finding that the IFN-γ response of NK cells to influenza A virus is dependent on the preexisting memory T cells specific for influenza A virus (22
), we propose that the enhanced NK cell reactivity to influenza A virus is secondary to the enhanced T-cell response after vaccination. In agreement with this hypothesis, we found that the change (n
-fold) in the percentage of IFN-γ+
NK cells correlated positively with the change (n
-fold) in IFN-γ+
T cells in both adults and children receiving either LAIV or TIV (data not shown).
Although the adults as a group did not respond to TIV or LAIV as assessed by a change in the average levels of influenza A virus-reactive IFN-γ+ cells, the responses were highly variable among individual subjects, who showed either increases or decreases in the percentage of IFN-γ+ T cells and NK cells after immunization. Significant positive and negative correlations were observed between measured parameters, indicating that such person-to-person variation reflects the intrinsic interaction between the vaccine and the host immune system rather than the measurement error of the assay. One of the most intriguing findings of this study was that in adults, who are likely to differ in their previous exposures to influenza A virus, CD4 and CD8 T-cell responses to vaccination best correlated inversely with the levels of CD56dim NK cell reactivity at the time of vaccination. To our knowledge, this is the first report of the relationship between the baseline levels of an innate immune response against a virus and the cellular immune responses after vaccination. The findings in this study, together with our previous findings on interactions between influenza A virus-reactive T cells and NK cells, suggest a new model of relationships between innate immunity and adaptive immunity, as well as between preexisting memory T cells and antigen recall responses, in the contexts of viral infection and vaccination.
Previously, we showed that IFN-γ production by NK cells in response to influenza A virus depends on preexisting influenza A virus-specific memory T cells and that interleukin-2 produced by the activated influenza A virus-specific memory T cells determines the IFN-γ response of NK cells to influenza A virus (22
). Based on these observations and the inverse correlation between cellular responses to influenza vaccines and the baseline percentage of IFN-γ+
NK cells, we speculate that the interaction between NK cells and DCs is critical in determining the immunologic outcome of exposure to influenza vaccination or infection.
DCs play a central role in both innate and adaptive immunity. Influenza A virus-infected DCs are the major producers of type I IFN and interleukin-12, which activate NK cells to produce cytokines and mediate cytotoxic activity (6
). On the other hand, DCs process viral antigens and present them to specific T cells, resulting in activation and amplification of virus-specific T cells, which constitute the primary or secondary T-cell response to viral infection or vaccination. Depending on their maturity and functional status, DCs can also render T cells anergic to cognate antigens (2
). Studies using in vitro-cultured NK cells and DCs have shown that NK cell-DC interaction may result in their reciprocal activation, as well as inhibition of DCs by NK cells, in different circumstances (16
). At low NK cell/DC ratios, DC responses were amplified dramatically, while at high NK cell/DC ratios, DC responses were inhibited completely. The inhibition of DC functions by NK cells was mediated by the potent DC-killing activity of the autologous NK cells (41
), which is most likely mediated by the CD56dim
NK cell subset, which expressed high levels of perforin (39
). Therefore, depending on the magnitude of CD56dim
NK cell activity during the early stage of infection in vivo, the NK cell response could either enhance or suppress subsequent adaptive T-cell responses by activating or inhibiting DC responses.
Based on our observations, the levels of influenza A virus-specific memory T cells are low in some adults, consistent with less previous exposure to influenza A virus infection or vaccination and resulting in low levels of baseline NK cell reactivity to influenza A virus. During influenza A virus infection or LAIV vaccination, the lower levels of baseline T-cell and NK cell reactivity are likely to result in higher levels of viral replication, as well as higher levels of DC activity, which are likely to be associated with a more vigorous influenza A virus-specific T-cell response. In contrast, higher baseline levels of influenza A virus-specific T-cell and NK cell reactivities should limit virus replication upon reexposure to natural infection or vaccination and inhibit DC functions. The outcome would be to limit the further activation of influenza A virus-specific T cells.
The variable patterns in the immune responses that we observed in influenza vaccinees also raise important questions regarding the roles of innate and adaptive cellular immunity during natural infection with pathogenic influenza viruses. The current population may have limited cross-reactive neutralizing antibody activity but significant T-cell cross-reactivity against newly emerged epidemic or pandemic influenza strains, as neutralizing antibodies primarily target the highly variable HA and NA proteins while T-cell immunity may target more conserved structural and nonstructural proteins of the virus. Do modulation of DC functions and subsequent T-cell responses by high levels of NK reactivity, which is due to high levels of preexisting memory T cells, accelerate or delay the recovery of infected individuals? Since innate immune responses to infecting virus may occur well before the onset of clinical symptoms, it is difficult to study the interaction between DC, NK cell, and T-cell immunities at the earliest stages of natural infection, as opposed to vaccination. Our study suggests that LAIV immunization may serve as a valuable model to address these types of critical questions concerning host responses to influenza virus and other viral infections, since early events can be studied directly in relation to the time of exposure.