The association between declining cellular immune function and loss of influenza vaccine efficacy with aging is well documented but the underlying mechanism is poorly understood. Clearly, additional correlates of protection to that provided by antibody titers are needed for advances in vaccine technology and the development of more efficacious vaccines for the older adult population. Current influenza vaccines contain split virus particles and thus as killed viruses, are poor stimulators of the CTL response that is largely directed by epitopes derived from the internal proteins of influenza virus in humans. Probably because of CTL memory from prior exposure to natural influenza infection, a CTL response to vaccination can be observed as a change in multiple read-outs of the CTL response to influenza vaccination [17
] including GrzB activity in PBMC stimulated with live virus[12
]. Although both antibody and CTL responses to influenza vaccination may contribute to protection, these responses may not necessarily be correlated [20
The data presented shows that the ex vivo CTL response to vaccination measured in an assay of GrzB activity predicts susceptibility to influenza illness. This GrzB activity correlates with cytolytic activity in 51
Cr-release assays in influenza-stimulated PBMC [8
]. GrzB is co-expressed with perforin and is contained in most CD8+ T cells expressing the degranulation marker, CD107a, consistent with a cytolytic effector function. Our results also suggest that activated NK cells contribute to the GrzB activity measured; however, it has been previously shown that expression of IFN-γ and perforin by these activated NK cells is a T-cell dependent process [22
]. Thus, the total GrzB response to influenza that is derived from or depends on T cells may be important for protection against influenza. Consistent with the substantial increased risk of complicated influenza illness in older adults with CHF, there was a marked reduction in the proportion of CTL and NK cells responding to influenza virus stimulation.
Preliminary results suggest that the high proportion of GrzB+ CTL in unstimulated PBMC and the poor response to virus stimulation in the CHF group is due to an increase in terminally differentiated effector memory CTL. Further experiments are planned to determine the amount of GrzB activity in purified CTL and NK cell subsets of resting and virus-stimulated PBMC and whether these terminally differentiated CTL can produce active GrzB In response to virus stimulation.
Previous studies in humans included only non-influenza-infected lung tissue. Thus, the question of function and differentiation of T cells can be answered at the site of but not at the time of influenza challenge. These studies in human lung tissue have shown much higher frequencies of influenza virus-specific memory CD8 T cells compared to peripheral blood. However, these lung resident CTL had a relatively late differentiation phenotype and low levels of GrzB compared to the higher levels expressed in virus-specific T cells in peripheral blood [23
]. The authors concluded that these lung resident CTL were non-cytotoxic memory cells. Our results are consistent with the findings of this study showing that memory CTL are in the peripheral blood rather than the lungs. In contrast to the CTL response to viral peptides alone [24
], we have shown that T cells responding to live influenza virus express high levels of GrzB and significantly increase the expression of CD107a on the cell surface, consistent with the development of a cytolytic effector function in the early stage of infection.
Unlike antibody responses to split-virus vaccines that are largely strain-specific, CTL responses are cross-reactive within most strains of influenza A (or within the strains of influenza B) and CTL memory can be restimulated by exposure to the virus. The data presented shows that older adult CTL have the capacity to mount an enhanced response to influenza vaccination, in this case due to restimulation of CTL memory from natural influenza A/H3N2 infection in the previous year. This is a very important observation suggesting that a reformulated influenza vaccine designed to stimulate the CTL response could improve protection in this population.
An important new development in the GrzB assay is the standardization of activity against a commercially available GrzB standard. The assay can now be validated across multiple laboratories and used to establish a threshold level of GrzB activity in ex vivo PBMC lysates as a correlate of protection. As an example of how this might be applied, it is important to understand that a correlate of protection can only be defined in those subjects with known infection, i.e., those who developed influenza illness. From this study, a protective level could be defined as GrzB activity greater than the upper limit of the 95% confidence interval at 4-weeks post-vaccination in the subset of subjects who subsequently developed influenza illness (200 U/mg protein). Future studies are designed to validate the assay in multiple laboratories and prospectively test this threshold of GrzB activity as a correlate of protection and consistency with current estimates of influenza vaccine efficacy and effectiveness in older people [25
In two separate influenza seasons, it has been shown that antibody titers do not effectively distinguish those older adults who will go on to develop influenza from those who will not. These results are consistent with an earlier study reporting that of 72 vaccinated elderly who were later confirmed to have influenza infection, 60% should have been protected based on classical hemagglutination inhibition (HI) assays (HI titer ≥ 1:40), with 31% of infected vaccinees having very high titers (HI titres ≥ 1:640) [27
]. Together, these data indicate antibody titers are a correlate, not a guarantee of protection; there are apparent successes and apparent failures of inactivated vaccine in older adults that cannot be explained by serum hemagglutination inhibition antibody titers [28
Poor vaccine-mediated protection in older adults has been attributed to age-related defects in cellular immune function based on polyclonal stimulation, which correlate with protective versus non-protective levels of influenza-specific antibody titers [29
]. Further studies combining measures of both humoral and cellular immune responses could shed light on how each of these responding lymphocyte subsets may contribute to cytolytic mechanisms of protection against influenza, and to cytokine-mediated cellular immune responses to influenza vaccination with aging [3
A potential limitation of this study relates to differences in the timing of the influenza seasons, starting in late November in the first year and in late January in the second year. However, the level of immune markers following vaccination appears to have been the same for both the early and later influenza season, and is consistent with previous results showing that the CTL response to influenza vaccination is maintained for 14–16 weeks following vaccination in this population [17
]. A second limitation is that influenza attack rates were too low to detect in subjects who developed influenza, a difference in GrzB levels between older adults with and without CHF. Although subjects with significant CHF (walking less than 1100 ft on the 6-MWT) have a slightly increased GrzB levels in influenza-stimulated PBMC, our flow cytometric studies suggest that this activity may be present at baseline and is associated with a poor response to influenza challenge.
In summary, our results suggest that the assay of GrzB activity in ex vivo virus-stimulated PBMC provides a responsive and complementary measure to antibody titers in the evaluation of influenza vaccine effectiveness in older adults. Further studies are needed to define a threshold level of GrzB activity as a correlate of protection in the development of both seasonal and pandemic influenza vaccines. Finally, we have shown that in spite of age-related changes that compromise immune function, older adults may still have to capacity to respond to reformulated influenza vaccines that are targeted to effectively re-stimulate CTL memory.