Responses to vaccination, such as the yearly influenza vaccine, are dramatically reduced in the elderly. A review of published studies over the last 20 years describes significantly lower levels of both seroconversion and seroprotection in elderly populations following influenza vaccination when compared with young subjects (32
). This is problematic since the elderly are much more susceptible to infections and are often targeted for vaccination. The immune response to vaccination can be enhanced by the use of adjuvants, but currently aluminum-containing adjuvants, such as alum, are the predominant adjuvant found in human vaccines. Alum induces the production of IL-4, IL-5, and IL-10 as well as the chemokines CCL2 (MCP-1), CXCL1 (KC), and CCL11 (eotaxin 1) by responding Ag-specific CD4 T cells (33
), inducing a vigorous type 2 immune response. Although Th2 effector generation proceeds efficiently in the young, our studies have shown that naive CD4 T cells from aged mice do not differentiate well to Th2 effectors without the addition of exogenous IL-2 (34
). Thus, the response to vaccination with alum-adjuvanted vaccines in the aged is dramatically reduced, as we have shown in this current study.
One straightforward approach to enhancing vaccine efficacy is to use more potent adjuvants. Published studies have demonstrated that the use of TLR ligands as adjuvants can significantly enhance the efficacy of vaccination in aged mice (35
). Moreover, we show here that the addition of poly(I:C) to our vaccine preparation could enhance the cognate helper activity of aged CD4 T cells, leading to a more robust humoral response. The use of specific TLR agonists may play a role in CD4 T cell differentiation. Poly(I:C) binds to TLR3 which is found predominantly on myeloid dendritic cells and induces activation of NF-κB and the production of high levels of PI cytokines, especially TNF-α and IL-6 (37
). These conditions could favor generation of a Th2- and/or Th17-polarized response, which would be ideal for generation of protective Ab following vaccination. In contrast, other TLR agonists may influence the T cell response differently than poly(I:C). For example, CpG oligonucleotides bind TLR9, which is more highly expressed on plasmacytoid dendritic cells, and mainly induces production of type I IFN and IL-12 (38
), which would drive generation of a more Th1-polarized response. Thus, by choosing TLR agonists based on the cellular expression of their receptor and the subsequent induction of cytokine production, it may be possible to direct the polarization of the CD4 T cell response.
To determine whether the induction of PI cytokines by TLR-binding adjuvants plays a role in vaccine efficacy, we examined the adjuvant effect of these cytokines directly in our adoptive transfer model. The use of the PI cytokine adjuvant in our studies is meant to be a proof of principle and assists in the determination of the mechanisms of action of the TLR agonists. The addition of PI cytokines to a vaccine preparation significantly enhanced the cognate helper activity of both young and aged CD4 T cells in young hosts (). In addition, the use of PI cytokines also resulted in an increase in all IgG isotypes as well as an increase in the levels of VH SHM in the presence of aged CD4 T cells. Thus, not only was the quantity of the humoral response enhanced (better B cell expansion and differentiation, higher IgG titers), the quality was also significantly enhanced (increased affinity maturation). Interestingly, although the addition of PI cytokines dramatically enhances the expansion of NP-specific cells when young donor T cells provide help, the titers of NP-specific IgG1 and the frequency of SHM remain unchanged (). This could be due to a kinetics issue (we only examined one time point for this study) or it could indicate that these two aspects of the B cell response are controlled by different mechanisms. Further studies to examine these points are planned.
We have gone on to show that these PI cytokines induce increased expression of the transcription factor RORγt and production of IL-17 family cytokines (IL-17, IL-21, IL-22) by both young and aged CD4 T cell populations. Importantly, we have also shown that in vitro-generated young and aged Th17 effectors have potent in vivo cognate helper activity. Thus, the ability of naive CD4 T cells from aged donors to differentiate into highly functional Th17 effectors remains intact even though they lose the ability to differentiate well toward a Th2 phenotype (34
Recently, IL-17 family cytokines, particularly IL-17 and IL-21, have been implicated in Tfh cognate helper function and the subsequent development of GC and plasma cells (39
). IL-21 impacts both CD4 T cell and B cell responses following immunization. In an autocrine feedback loop, Tfh cells produce IL-21 and also express the IL-21 receptor, which drives expression of CXCR5 and responsiveness to CXCL13 (42
). In addition, IL-21 was shown to also promote B cell activation, Ab production, and plasma cell differentiation (40
). Although it is true that IL-17 production results from inflammation (IL-6 in particular) and IL-17 has been shown to be involved in induction of autoimmunity (43
), its critical role in the protection from infection is just beginning to be elucidated. IL-17 production can recruit neutrophils to infection sites (44
) and Th17 effectors can enhance Th1 responses. Importantly, Th17 can also recruit Th1 effectors to these sites of infection (30
), leading to a more robust antibacterial response. Mice that lack IL-17 expression also exhibit significantly reduced CD4 T cell-dependent Ab production (45
), demonstrating the importance of this cytokine in humoral responses. In addition, Th17 effectors can produce IL-21, which is also important for generation of Ab to influenza as shown in our recent publication (46
). Thus, much like IFN-γ, IL-17 can be involved in both autoimmune responses and responses to infection and vaccination.
IL-17 produced by responding CD4 T cells drives the enhanced frequency and duration of GC development by modulating the effects of chemokines, especially CXCL12, which is involved in B cell migration and retention (47
). Although Nurieva et al. (41
) demonstrated that Tfh do not absolutely require TGF-β or RORγt and, thus, IL-17 for their differentiation to functional helper cells, this does not mean that IL-17-producing CD4 helper T cell subsets cannot also provide B cell help. As our results in this study demonstrate, the Tfh subset is likely to be quite heterogeneous and it may actually describe a functional subset rather than a specific differentiated subset. This has been elegantly demonstrated in a recent study showing that the immune response to a successful vaccination is very broad, complex, and highly integrated (48
). This report showed that following immunization, a complex innate immune response is initiated, resulting in a variety of Th cytokine-producing subsets, including Th1 and Th2. These Th subsets exhibit an array of protective functions, including cognate help for B cells and induction of robust Ab production. Importantly, our studies demonstrate that by using inflammatory adjuvants, we can drive the differentiation of naive CD4 T cells from aged animals into potent helper T cells and generate a robust humoral response. We suggest that by using inflammatory adjuvants, we have broadened the response of the Ag-specific CD4 T cells and, thus, have enhanced their ability to function in vivo. Most notably, by enhancing production of IL-4, IL-17, and IL-21, our immunization protocol with PI cytokines has enhanced the differentiation of follicular helper T cells, even from aged naive CD4 T cells. Ultimately, this could result in enhanced efficacy of vaccinations for the elderly.