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Experimental Gerontology 42 (2007) 438–440

Mini Review

How vaccines work on the background of the aging immune system

Laura Haynes *

Trudeau Institute, 154 Algonquin Avenue, Saranac Lake, NY 12983, USA

Received 25 September 2006; accepted 9 October 2006Available online 27 November 2006

Abstract

The elderly exhibit reduced responses to vaccinations, leaving them more susceptible to preventable infectious diseases such as influ-enza and pneumonia. It has been unclear as to the specific changes in the aging immune system that contribute to this decline, thus wehave developed a mouse model to examine this phenomenon and determine why it occurs and how it can be overcome. Our ultimate goalis to determine how to enhance vaccine efficacy for elderly populations.� 2006 Elsevier Inc. All rights reserved.

Keywords: Aging immune system; Vaccinations; CD4 T cells

When the aged are vaccinated, the resulting antibodytiters are lower than those seen in younger individualsand the function of the antibodies produced is also dimin-ished (i.e. affinity maturation of the antibodies is reduced)(Miller and Kelsoe, 1995; Zheng et al., 1997). Our studieshave addressed this issue by developing a mouse modelto determine which aspects of the immune responses areinfluenced by aging. The majority of these results have beenpublished within the past two years (Eaton et al., 2004;Haynes and Eaton, 2005; Haynes et al., 2004). The modelemployed for these studies involves immunization with ahaptenated protein: nitrophenyl conjugated to pigeon cyto-chrome c (NP-PCC). This model is very useful for thesestudies since it allows for the examination of NP-specificB cells by flow cytometry. Responding B cells can be iden-tified by the use of NP conjugated to a fluorochrome andthe expansion and cell surface phenotype of the respondingB cells can be easily determined and quantitated. Uponimmunization of mice with NP-PCC/alum, NP-specific Bcells expand and differentiate to a germinal center (GC)phenotype (CD38loPNAhi). They also undergo classswitching from IgM to IgG (IgG1 when alum is used),which can also be examined using flow cytometry. Impor-

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doi:10.1016/j.exger.2006.10.012

* Tel.: +1 518 891 3080; fax: +1 518 891 5126.E-mail address: lhaynes@trudeauinstitute.org

tantly for our studies, this response is highly dependentupon the helper activity of CD4 T cells.

When aged B10.BR mice were immunized with NP-PCC, the expansion of NP-specific B cell populations andthe production of NP-specific IgG were significantlydecreased compared to young (Table 1). To specificallyexamine the effects of age on the function of lymphoid cellsinvolved in this response, we developed an adoptive trans-fer model as shown in Fig. 1. In this model, PCC-specificCD4 T cells from young or aged T cell receptor transgenic(TCR Tg) mice were transferred into syngeneic young oraged CD4KO mice. By using CD4KO mice, which haveno endogenous helper cells, the in vivo helper activity ofthe donor CD4 T cells can be easily assessed. The advanta-ges of this model are that it allows a direct comparison ofequal numbers of naive antigen specific CD4 T cells fromyoung and aged donors and it also allows us to examinethe influence of aging on host and donor CD4 T cell func-tion separately.

When young and aged donor TCR Tg CD4 T cells weretransferred into young CD4KO hosts immunized with NP-PCC/alum, the humoral responses in hosts receiving ageddonor cells was significantly reduced (Table 1). There wasreduced expansion of the NP-specific B cell populationand reduced differentiation of NP-specific B cells to a ger-minal center phenotype (CD38loPNAhi). There was alsoreduced NP-specific IgG1 production in the host mice

Table 1Summary of NP-specific B cell responses in intact mice and in adoptive transfer model

Intact miced Young hosts Young CD4

Y A Y CD4 A CD4 Y host A host

B cell expansiona ++++ ++ ++++ ++ ++++ ++++GC differentiationb ++++ + ++++ + ++++ ++++Serum IgGc ++++ ++ ++++ ++ ++++ ++++

a Expansion of NP-specific B cell population following immunization.b Differentiation of NP-specific B cells to GC phenotype CD38loPNAhi.c Serum titers of NP-specific IgG1.d Responses measured in intact young and aged B10.BR mice and in adoptive transfer model shown in Fig. 1.

Young or AgedAND TCR Tg

naive TCR TgCD4 cells

Transfer intomice lackingendogenous

T cells

Young or AgedCD4KO hosts

Assay for T celland B cellresponses

VaccinateNP-PCC

Culture with peptide Ag/APC

+/- cytokines

Fig. 1. Adoptive transfer model for examining CD4 cognate function.Naive CD4 T cells were harvested from young and aged AND TCR Tgmice. They were either transferred into CD4KO hosts or cultured for 4days with Ag/APC ± exogenous cytokines to generate effector popula-tions. These effector populations were then transferred into CD4KO hosts.Young or aged adoptive hosts were immunized with NP-PCC/al-um ± exogenous cytokines. On days 7, 14 and 21 post-immunization,the expansion and differentiation of the NP-specific B cells was examinedusing flow cytometry. The titers of NP-specific IgM and IgG in serum werealso examined.

Table 2Effect of cytokines on the helper function of aged CD4 T cell effectorpopulationsa

No cytokines IL-2 T16

B cell expansionb ++ ++ ++++GC differentiation ++ ++ ++++Serum IgG ++ ++ ++++

a Effectors were generated with Ag/APC and the indicated cytokines for4 days. Effectors were then transferred into young CD4KO hosts as shownin Fig. 1.

b Expansion and differentiation of NP-specific B cells and NP-specificIgG1 production determined as in Table 1.

L. Haynes / Experimental Gerontology 42 (2007) 438–440 439

which received the aged CD4 T cells. There was no differ-ence in the ability of the young and aged donor cells tomigrate into B cell follicles and germinal centers, but thegerminal centers that were formed in the presence of agedCD4 T cells appear more disorganized compared to thoseformed when young T cells were present. While there wasno difference in expression of several cell surface moleculesinvolved in cognate helper activity, such as CXCR5,CD134 and CD28, the aged CD4 T cells did express lowerlevels of CD154 following antigenic stimulation, whichcould significantly impair their ability to provided help toB cells.

To examine the effect of host age on humoral responses,we used the same model but transferred young Tg CD4 Tcells into young or aged CD4KO hosts. Upon immuniza-tion, the response of the NP-specific B cells was not differ-ent in the young and aged hosts receiving young CD4 Tcells (Table 1). The expansion and germinal center differen-tiation was similar as was the production of serum IgG.There was also very similar priming of the donor T cellsin both the young and aged hosts, indicating little effect

of aging on antigen presenting cells. Thus, in this model,age has a dramatic effect on CD4 T cell function but haslittle effect on B cell responses.

The next series of studies examined ways to enhance thecognate helper activity of aged TCR Tg CD4 T cells. Effec-tors were generated from young and aged naive TCR TgCD4 T cells in vitro with antigen and antigen presentingcells (Ag/APC) with or without added cytokines. Effectorpopulations were then transferred to young CD4KO hostsas in Fig. 1. When young and aged effectors were generatedwith Ag/APC alone, there were significant defects in thehelper function of the aged effectors, similar to aged naiveCD4 T cells. Since our earlier studies showed that additionof interleukin-2 (IL-2) could enhance the in vitro prolifera-tion and differentiation of aged CD4 T cells (Haynes et al.,1999), we tested this cytokine for the ability to enhancehelper activity. Young and aged effectors were generatedin the presence of Ag/APC and IL-2 for 4 days and thentransferred into CD4KO hosts immunized with NP-PCC/alum. While IL-2 does enhance the in vitro response ofaged cells, the cognate function of aged IL-2 effectorswas not enhanced and a significant reduction in NP-specificB cell responses was still observed (Table 2).

Our previous studies have also shown that a combina-tion of the cytokines Tumor Necrosis Factor a (TNFa),IL-1 and IL-6 (T16) could enhance the in vitro and in vivoproliferation and IL-2 production by aged CD4 T cells.Thus, we next examined the ability of these cytokines toenhance the cognate function of aged T cells. Four dayeffectors generated with Ag/APC and T16 were transferredto CD4KO hosts immunized with NP-PCC/alum. In thiscase, we found that young and aged effectors exhibited sim-

440 L. Haynes / Experimental Gerontology 42 (2007) 438–440

ilar robust cognate helper activity. The expansion and dif-ferentiation of NP-specific B cells as well as the productionof serum antibody was similar, whether young or agedeffectors provided help (Table 2). Not only was the produc-tion of NP-specific IgG1 enhanced, other IgG isotypes(IgG2a, IgG2b, IgG3) were also enhanced upon the useof these cytokines. The same results were also found whenthe cytokine cocktail was used directly as an adjuvant.Thus, the combination of the cytokines T16 dramaticallyenhanced the cognate helper function of aged CD4 T cells.

Based on our studies, we propose that adjuvants thatcan induce the production of T16 in aged animals (orhumans) should be tested for their ability to enhance vac-cine efficacy. Our studies would predict that the use of suchadjuvants would enhance antibody production and, thus,enable elderly persons to be better protected from infec-tious diseases such as influenza.

References

Eaton, S.M., Burns, E.M., Kusser, K., Randall, T.D., Haynes, L.,2004. Age-related defects in CD4 T cell cognate helper functionlead to reductions in humoral responses. J. Exp. Med. 200,1613–1622.

Haynes, L., Eaton, S.M., 2005. The effect of age on the cognate functionof CD4+ T cells. Immunol. Rev. 205, 220–228.

Haynes, L., Eaton, S.M., Burns, E.M., Rincon, M., Swain, S.L., 2004.Inflammatory cytokines overcome age-related defects in CD4 T cellresponses in vivo. J. Immunol. 172, 5194–5199.

Haynes, L., Linton, P.-J., Eaton, S.M., Tonkonogy, S.L., Swain, S.L.,1999. IL-2, but not other common c chain cc)-binding cytokines, canreverse the defect in generation of CD4 effector T cells from naive Tcells of aged mice. J. Exp. Med. 190, 1013–1023.

Miller, C., Kelsoe, G., 1995. Ig VH hypermutation is absent inthe germinal centers of aged mice. J. Immunol. 155, 3377–3384.

Zheng, B., Han, S., Takahashi, Y., Kelsoe, G., 1997. Immunosenescenceand germinal center reaction. Immunol. Rev. 160, 63–77.