3.1. Cross-reactivity of the T cell response
In contrast to the strain-specific antibody response of B-cells, viral epitopes that stimulate T helper cells (Th) and cytotoxic T lymphocytes (CTL) responses are more conserved across different strains of influenza and appear not to degrade with antigenic drift (51
). Virus is taken up and processed by antigen-presenting cells (APC) such as macrophages and dendritic cells, and the resulting peptides are presented with the major histocompatability complex (MHC) to activate T-cells (54
). Protein sequences within HA and NA and internal viral proteins (matrix and nucleoproteins) that stimulate Th (CD4+) and CTL (CD8+) responses have been identified. Importantly, these peptide epitopes are conserved across subtypes of influenza A (A/H3N2 vs. A/H1N1), and between types of influenza (influenza A vs B)(55
), and are cross-reactive between the different strains of influenza. Current killed virus vaccines are poor stimulators of the CTL response (discussed below) and thus are not optimal for stimulating CTL which are necessary for clearing virus once infection occurs. Our work and others have shown that defects in cell-mediated immune responses are associated with increased risk for influenza illness in older adults and that such change cannot be detected by the antibody response alone to influenza vaccination (28
illustrates the “in vivo” response to influenza vaccination wherein vaccine is taken up by dendritic cells or macrophages (antigen-presenting cells, APC) at the site of vaccination. Inflammation stimulated by the injection and potentially enhanced by adjuvant facilitates the activation of the APC and uptake of the virus. The APC then migrates to the local lymph node to interact with B cells, T helper cells (Th) and cytotoxic T lymphocytes (CTL). Presentation of vaccine-derived peptides on the Major Histocompatibility Complexes, MHC I and MHC II, drives the T cell response to stimulate B cells for antibody production and produce a variety of cytokines and cytolytic mediators, as will be discussed. Most importantly, this interaction stimulates T cell memory that can be activated during a subsequent exposure to natural infection to protect against the development of influenza illness and its serious complications. Antibodies are generated through T-cell dependent mechanisms and their production can be stimulated by a Th1 or Th2 response to vaccination. Antibodies mediate protection through virus neutralization on the mucosal surface and hemaglutination inhibition of virus attachment to the epithelial cell to prevent infection, or through NK-mediated cytotoxity or complement-mediated lysis that are dependent on antibody binding to virus-infected cells.
The response to vaccination is summarized (left diagram) and the response to virus stimulation in PBMC cultures is illustrated (right diagram).
Ex vivo stimulation of peripheral blood mononuclear cells (PBMC) simulates the activation of the T cell response during natural infection. In this case, uptake of live virus results in effective presentation of viral peptides on both MHC I and MHC II, respectively activating CTL and Th1/Th2. Natural infection in the lungs activates Th1/Th2 in the adjacent lymph nodes and the resulting cytokine response may be regulated to favor a Th1 or Th2 response. A Th1 response is needed for effective activation of the memory CTL needed to clear virus from the lungs. The magnitude of the T cell response to natural influenza infection is simulated in ex vivo PBMC by measuring cytokines expressed in T cells, by flow cytometry, or levels in the culture supernatants, and the cytolytic function or activity (granzyme B) in lysates of stimulated PBMC.
3.2. The role of T helper cells in the response to influenza
It has been well documented that T-cell-mediated immune responses are also protective against influenza infection and illness (56
). Th (T helper cell)-mediated immune responses to influenza virus play a key role in both humoral and CTL responses to influenza vaccination. T helper type 1 (Th1) effectively stimulate antibody responses, IFN-γ production, and CTL memory, while Th2 stimulate antibody responses and IL-10 production, which suppresses the Th1 response. Anti-influenza antibodies are stimulated by Th1 and Th2 to respectively produce IgG2a and IgG1 responses to influenza vaccination. With aging, a decline in Th1 relative to Th2 cytokine production occurs. Also important to vaccine development, Th and CTL recognize viral peptides on MHC II and MHC I, respectively, and thus have different requirements for effective antigen presentation. Peptides derived from replicating live virus are effectively presented on both MHC I and MHC II. Split-virus vaccines are killed viruses and are effectively presented on MHCII but presentation on MHC I requires antigen cross-presentation within the dendritic cell, and generally stimulate a weak CTL response (57
) that depends on previous priming from natural infection (58
). Human studies have confirmed that CTL responses effectively clear influenza virus even in the absence of protective antibodies to the infecting virus, and are important for recovery from influenza infection (59
). Of note, recruitment of CTL into infected lung tissue is dependent on Th1 cytokine production and thus the shift from Th1 to Th2 cytokine production may further compromise the immunologic defense mechanisms against influenza in older adults. These changes are reflected in the finding that a reduced IFN-γ:IL-10 ratio and lower levels of cytolytic mediators in PBMC responding to influenza challenge correlates with risk for influenza illness in older adults (32
). Thus, influenza vaccines that could produce a shift toward a Th1 response and more effectively stimulate both antibody production and CTL memory, would improve protection in older adults. These are the types of vaccines that are needed for improved protection in older adults. However, it should noted be that the formulation of the vaccine is also important as the delivery of viral antigens for MHC Class I presentation depends on cross-presentation of antigens derived from the vaccine. Thus, the induction of Th1 cytokines alone is insufficient for stimulating a CD8 T cell memory response to the vaccine. Further, CD8 T cell responses are directed against internal components of the viruses and, hence, provide cross-protection. However, influenza vaccines that are currently marketed in the US and Canada vary in the amount of internal proteins (60
) and may vary in their ability to recall or induce CD8 T cells, even if they are formulated for Class I processing and presentation.
3.3. Immunosenescence: The loss of T-cell adaptive immune function impacts on vaccine responsiveness
Thymic involution and a decline in naïve T cell output with increasing age, together with a lifetime of exposure to a variety of pathogens, leads to a dramatic reduction in the naïve T cell pool and a relative increase in the proportion of memory T cells. While the reduction in naïve T cells with aging may be important to overall immunity, prior exposure to influenza and the generation of memory T cells may mean that function in this T cell compartment is most important for protection against influenza. Within the total memory pool, the most dramatic functional changes occur in the CD8+ T cell subset; progressive exhaustion of this compartment leads to the loss of costimulatory molecules (CD28), shortening of telomeres, and terminal differentiation to end stage cells that are resistant to the usual apoptotic mechanisms that control the size of memory T cell clones responding to a particular pathogen (61
). It has been shown that most of these CD8+CD28- memory T cells are part of large clonal expansions specific for persistent viruses, mainly CMV (62
), but also Epstein-Barr virus (EBV) and varicella zoster virus (VZV) (63
). Although these viruses typically establish asymptomatic latent infection with intermittent subclinical episodes of reactivation, suppression of disease activity is related to CD8+ T lymphocyte presence and function. By old age, excessive accumulation of these virus-specific CD8+ T lymphocytes eventually overgrows the T lymphocyte pool, compromising immune function and restricting the overall immune repertoire (64
). Because the Th and CTL responses are cross-reactive between the different strains of influenza A or influenza B, memory from previous exposure to the virus is re-stimulated with vaccination. Thus, the impact of age-related changes in memory T subsets is likely to have the greatest impact on the response to influenza vaccination and protection from influenza illness.
3.4. Age-related changes in T cells are much greater in CD8+ relative to CD4+ subsets
In contrast to what has been observed within the CD8+ T cell population during aging, CD4+ T cells are relatively less affected by replicative senescence. Indeed, CD28 expression is preserved within this subset during aging (65
). Although a normal CD4+ T cell response to influenza vaccination is observed in older adults, over the long-term, the memory CD4+ T cell response is impaired (66
). These long-term memory changes do not appear to affect the duration of the serum antibody response to influenza vaccination, which is similar in young and older adults (67
), but may contribute to dysregulated cytokine responses that promote virus replication once infection occurs.
3.5. Age-related increase in inflammatory cytokines and dysregulation of T-cell cytokines
Cultures of senescent CD8+ T lymphocytes produce high levels of certain pro-inflammatory cytokines, such as TNFα and IL-6 (68
), cytokines that are associated with frailty (69
). Thus, CD8+ T cells may directly contribute to elevated serum levels of inflammatory cytokines (IL-1, IL-6, TNF-α) causing dysregulation of the cell-mediated immune response (70
). While a cocktail of TNF-α, IL-1 and IL-6 has been shown in mouse splenocytes to reverse age-related changes in cognate helper T cell function for antibody production (71
), we have shown that this inflammatory cytokine cocktail suppresses the CTL response to influenza virus (unpublished observations). A greater understanding is needed as to how with aging, T helper cell production of cytokines is dysregulated and defects in the CTL response to influenza contribute to loss of vaccine-mediated protection. These studies are critical to development of influenza vaccines with improved clinical protection against serious disease in older people, and will complement the antibody responses, which are currently the only available measure of influenza vaccine efficacy in clinical trials.
3.6. T helper cytokine regulation impacts on the response to influenza in older adults
The Th-mediated immune response to influenza virus plays a key role in the generation of both humoral and CTL responses to influenza vaccination. Previously, Th1 and Th2 were defined by their cytokine products such that the Th1 cytokine, IFN-γ, down-regulated Th2, and IL-10 down-regulated Th1 (72
). While this paradigm is generally applicable in the mouse model, recent studies have questioned the validity of the Th1/Th2 paradigm in humans, and the contributions of regulatory T cells (Treg) and Th17 subsets to cytokine regulation are only beginning to be understood (75
). Under a revised model, naïve CD4+ helper T cells are stimulated by IL-12 to produce IFN-γ i.e. become Th1);IL -4 stimulates Th2 to produce IL-4, IL-5, IL-10, IL-13; and IL-1, IL-6 and IL-23 to stimulate Th17 to produce IL-17, IL-22 and IL-26. Treg produce IL-10 and TGF-β, which downregulate inflammatory responses including the production of cytokines such as IFN-γ and IL-6. All of these Th subsets have counter-regulatory interactions between each other (76
) but the impact of aging on the dysregulation of these interactions is only beginning to be understood.
Our data has shown that the IFN-γ:IL-10 ratio correlates with risk for influenza illness (32
) but characteristics of the vaccine recipient and PBMC culture conditions may alter this relationship (77
). The apparent downregulation of IFN-γ by IL-10 may be Th2 or Treg-mediated (81
), and the interaction with Th17 may also be important. Studies in human PBMC show that Th17 promotes the recruitment of IFN-
producing T cells and as such, is regulated by the tissue level of IFN-γ (82
). While IFN-γ appears to be important in immune defense mechanisms against influenza, aberrant IL-17A production has been shown to stimulate a neutrophil-dependent increases in pro-inflammatory cytokines in response to systemic viral infection that contributes to death in aged mice (83
). Further, we have found that from a panel of Th1/Th2/Treg/Th17 cytokines, IL-17 is the only cytokine that showed an age-dependent increase in the cytokine response to live influenza challenge while all other cytokines declined with age (unpublished observations). Since IL-2 has been shown to constrain the formation of Th17, the decline in the IL-2 response with aging may explain the increase in IL-17 in influenza-stimulated PBMC. Further, age-related increases in IL-17 responses to influenza virus may be relevant to poor outcomes in this population. The effect of vaccination on Th17 remains to be studied.
3.7 What are the likely cell-mediated immune correlates of protection against influenza?
Th and CTL have different requirements for effective presentation of viral peptides on MHC II and MHC I, respectively (57
). Because only Th and B-cells are effectively stimulated by killed virus (as in the trivalent inactivated split virus influenza vaccine), vaccination stimulates good antibody responses, but only weak CTL responses in adults; the CTL response most likely results from restimulation of a previously primed response to influenza through natural infection (58
). Human studies have confirmed that CTL responses are important for recovery from influenza infection even in the absence of protective antibodies to the infecting virus strain (59
). CTL combat influenza infection by recognizing MHC I-viral peptide complexes on virus-infected host cells, and destroy them before infective progeny virus can be released (85
). A direct comparison showed that protection correlates with the virus-specific CTL (CD8+) response in the lungs and associated lymphoid organs. Although self-renewing populations of virus-specific CD8+ T cells are maintained for many years after influenza infection, protective cellular immunity is short-lived and disappears within six months (90
). We have demonstrated that memory CTL from natural infection may be restimulated by influenza vaccination (33
) and demonstrated the potential for older adults to mount an enhanced CTL response to vaccination. Novel vaccines designed to stimulate this enhanced response could be more effective in older adults.
3.8. The key function of granzyme B in CTL-mediated Apoptosis
Virus-specific killing is mediated by granzymes contained in granules within CTL. Granules containing granzymes and perforin migrate to the “immune synapse” between the activated CTL and the virus-infected target cell. Granzymes are transported across the cell membrane by perforin into the cytoplasm of the target cell, and granzyme B (GrzB) is involved in an enzymatic cascade that leads to apoptotic cell death of the virus-infected cell (93
). GrzB is a key element of the T-cell response to influenza in the lung (94
). The development and validation of the GrzB assay (96
), which correlates with cytolytic activity by standard 51
Cr-release assays (97
), quantifies the amount of GrzB activity and complements measures of influenza-specific CTL frequencies. Ex vivo studies have shown no difference in influenza-specific CTL frequencies between young and older adults (99
) and suggest that the defect in influenza susceptible older persons is the amount of GrzB produced on a per CTL basis. Importantly, ex vivo levels of GrzB in lysates of influenza-stimulated PBMC correlate with risk for influenza illness in our studies (32