We set out to test the prediction that adults not known to have been exposed to the pH1N1 influenza virus would have memory CD8+ T cells that cross-react to the novel strain as well as to recent and prototypic influenza strains. Our results support the hypothesis that CD8+ T cells previously primed with multiple strains of seasonal influenza virus are activated when stimulated with pandemic influenza virus. Single cytokine producing cells were by far the predominant type of CD8+ T cells observed, regardless of which virus was used to stimulate the cells. For a given individual, pattern of the cytokine responses were similar among all the viruses tested, suggesting that the novel pandemic strain is not unusual in the types of immune responses it elicits. TNFα was the most frequently observed cytokine, followed by IFNγ; IL2 single positive CD8+ T cells were not detectable above the negative controls. The low frequency of IL2 CD8+ T cells in the periphery may indicate a predominance of effector phenotype cells over memory phenotype in some subjects (29
The functional heterogeneity of the virus specific CD8+ T cells is further emphasized by the observation that some cells produced two or more cytokines. T cells that produced both TNFα and IFNγ had the highest prevalence, followed by those that produced IFNγ and IL2. Although single IL2 producing cells were not significantly more frequent among cells stimulated by virus than by controls, when IL2 positive cells were coupled with IFNγ alone or with IFNγ + TNFα, a virus-specific IL2-producing population could be discerned above the background levels. Findings recently reported by Makedonas et al, who observed a negative correlation between perforin upregulation and IL2 secretion (30
), would suggest that polyfunctionality associated with IL2 might distinguish a memory from effector phenotype. Our result in the context of their and other published findings suggests that cells responding with IL2 may represent a population poised to proliferate in response to brief antigen-stimulation prior to exerting additional antiviral effector functions (31
). IL2-producing CD8 T cells are also associated with better memory CD8 T cell formation (32
). However, the significance of the IL2 producing populations of CD8 T cells in our studies is not known.
Our finding of an expanded population of influenza-specific polyfunctional cross-reactive memory CD8+ T cells in subjects without recent exposure to influenza virus or vaccination is novel. Polyfunctional CD8+ T cells have been reported to be associated with disease control in the setting of chronic infections such as HIV and Hepatitis C (23
). It has also been postulated that polyfunctional T cells represent a highly active, less exhausted phenotype that may be more effective at controlling infection (35
) than single-cytokine-producing cells. Less evidence for the role of polyfunctional CD8+ T cells in acute infection is reported. Our finding of increased IFNγ MFI in association with IL2-secreting cells suggests that these may be a distinct population of highly active cells capable of proliferating and secreting cytokine with acute infection, but perhaps not exerting cytolytic function. The use of mean fluorescence intensity to compare relative size of cytokine output per cell has been validated (37
) and used to study T cells in autoimmunity and chronic disease, but has not been examined in influenza-specific T cells. This method may be a useful approach to comparing relative activity in in vitro
CD8+ T cell assays. Evaluating cytotoxic function of IL2+ polyfunctional cells may also aid in better defining the roles of CD8+ T cell populations.
Cross-protective adaptive immunity is important in antigenically dissimilar influenza viruses, as has been shown in animal studies and limited human studies (8
). There is strong evidence in the literature that CD8+ T cells play a large role in providing such cross-immunity to influenza virus that has drifted enough to evade neutralizing antibodies (13
). Jameson et al showed that in humans cytotoxic T cells specific for seasonal influenza epitopes showed cross-reactivity with avian and swine-origin strains. Degree of cytotoxicity to heterosubtypic challenge was similar to homosubtypic challenge, and was largely dependent on HLA-type and conservation of amino acid sequence within known CD8+ T cell epitopes.
The clinical relevance of CD8+ T cell function has been partially explored in two recent publications. Shahid et al showed that low granzyme B activity and low IFNγ:IL10 ratio pre-illness were correlated with susceptibility to influenza (49
). This correlation was first demonstrated by McElhaney et al in 2006, who measured serum antibody titers and PBMC granzyme and cytokine responses in elderly subjects immunized with inactivated influenza vaccine. They found that in this population, higher IFNγ:IL10 ratio correlated with protection, whereas antibody levels did not (50
). In their more recent publication, this same group showed a negative correlation between granzyme B activity and influenza infection (51
). The importance of T cells is also supported by experiments in animal models that show T cell-mediated protection from heterosubtypic influenza infection in the absence of B cell immunity (52
). With regards to the pH1N1 virus, our recent studies demonstrate substantial CD4 and CD8 T cell mediated protection from lethal infection in animals primed with laboratory or recent seasonal human influenza viruses, and no evidence of serological protection even with sera derived from sH1N1 primed donors (pH1N1 serum was fully protective)(53
). These studies highlight the importance of studying the quality, not simply the frequency, of T cell immunity in predicting a population’s susceptibility to disease. If this phenomenon were consistent with virus behavior in humans, it would be a potential explanation for disease attenuation in the setting of high susceptibility. Further studies are required to demonstrate any clinical benefit associated with polyfunctional CD8+ T cells, though if demonstrated, could serve as a novel immune correlate.
The results of this study utilizing whole virus in vitro
stimulation are consistent with findings of several other groups investigating pre-existing immunity to pH1N1 in humans. Greenbaum et al.
demonstrated detectable IFNγ secreting memory phenotype CD8+ T cells in response to peptide pools of conserved H1N1 epitopes (17
). Tu et al.
found a population of CD8+ T cells that had cytotoxic activity against pH1N1 and expansion of antigen-specific IFNγ secreting cells from subjects recently vaccinated for seasonal influenza. Though the frequency of antigen-specific cells was lower with pH1N1 stimulation than with seasonal influenza, the results were still statistically significant (47
Our study focused on analysis of cross-protective CD8+ T cell responses, our assay development revealed several important factors that enable the use of whole virus for in vitro
stimulation. These include the use of allantoic fluid as a negative control when utilizing egg-grown viral stock, and use of multiple viral preparations to account for variability in CD8+ T cell responses. We felt it was important to understand the CD8+T cell response to whole virus, since these cells are presumably reacting to processed virus from within infected antigen presenting cells. Use of whole virus most closely simulates natural infection and antigen presentation. Unfortunately, this approach does introduce some questions about how similar an individual lab viral strain is to the circulating influenza strain, and whether small differences in lab strains account for differences in results. Although we found a significant increase in cytokine-producing CD8+ T cells in response to pH1N1 as compared to negative controls, there was a consistent trend toward lower frequencies when compared to seasonal or laboratory influenza-specific responses. It is possible that changes in the surface proteins of influenza change the signals received by CD8+ T cells, thereby changing either proliferation or cytokine profile. At this time, however, whether differences between influenza strains result from a lower frequency of precursor T cells specific to pH1N1, or is intrinsic to the virus strains, is not clear. The effects of egg and tissue culture passage of human influenza viruses on human lymphocyte infectivity and antigen-presentation have not been exhaustively investigated. Such an investigation could shed some light on these issues. Interestingly, in animal studies, the A/California stock used in these studies shows a high degree of virulence (53
), similar to the A/PR/8/34 laboratory strain, consistent with the view that the virus is fit for replication in cells other than the highly permissive eggs and MDCK cell line. Nevertheless, it is possible that the variability in the HA protein introduced in the pandemic strain affected this virus’s ability to infect PBMC, which would explain not only the lower T cell response, but also its retained ability to cause disease in the respiratory tract.
Overall, we observed measureable but low frequencies of cross-reactive influenza-specific CD8+ T cells in the circulation of subjects who had not recently been infected. The majority of circulating memory T cells specific for influenza is in a quiescent state with restricted functionality. Our finding of rare polyfunctional cytokine producing cells suggests that protection from disease by these T cells may be quite limited. The finding of such few polyfunctional CD8+ T cells may partly explain the significant number of healthy young and middle-aged adults who exhibited signs of infection during the pandemic. Based on seroprevalence and exposure studies, Carrat et al estimated that asymptomatic H1N1 influenza infection may occur in around 30% of exposed individuals (54
), though exposure rates are difficult to accurately determine. It is therefore possible that either infrequent exposure or recurrent asymptomatic seasonal influenza infection may lead to a more terminally differentiated effector CD8 T cell population. This has been shown in models of either chronic active or latent infection, where frequent, repeated antigen exposure is associated with a more terminally differentiated phenotype, that is less likely to proliferate on cognate antigen engagement (27
). It will be interesting to see if the frequencies of polyfunctional CD8+ T cells are increased in subjects recovered from a documented infection.
Alternatively, it is well established that optimal immune protection from heterosubtypic infection is provided only when the memory T cells are located in the respiratory tissue (38
). Our present analysis did not specifically investigate the homing potential of the T cells, so we cannot draw any conclusions as to the relationship between cytokine production and homing.
We showed evidence of pre-existing CD8+ T cell immunity to pH1N1, however, it is not known to which epitopes these cells are responding. Immune models have predicted the likely presence of pre-existing immunity to pH1N1 based on the high percent of conserved MHC Class I epitopes (17
) found in bioinformatics resources. It would seem logical that as memory cells, CD8+ T cells are responding to epitopes highly conserved among different influenza viruses. Verification that the CD8+ T cells observed are reacting to conserved sequences will require careful mapping of class I HLA restricted epitopes. It is also possible that small nucleotide changes among the viruses lead to altered recognition in different individuals based on HLA type. Resolution of these unanswered questions will be critical in targeting CD8+ T cells in vaccine design. Our study does raise the question about how effective cytotoxic T cell immunity is in controlling a pandemic, however, since the subjects studied did fall in the demographic of high susceptibility.