Mouse model of age-related susceptibility to WNV
We established a robust mouse model of the age-related susceptibility to WNV. In our hands, old mice exhibited increased susceptibility to WNV regardless of the infection route (i.p. or s.c.), the viral isolate of WNV Ia (NY-99, 31A, or 385–99; Table S1
, ; see and ; and not depicted), or the mouse strain (, C57BL/6; and see and , C57BL/6; or Fig. S1
, BALB/c) used, although, as expected, i.p infection produced lethal effects at a lower dose (1–20 PFU) compared with the more physiological s.c. infection (50–1,000 PFU). Overall, old mice were at least six times more susceptible to WNV as measured by survival rates over many viral concentrations ( and not depicted). At low viral doses, that difference was drastically reduced and, in some experiments, disappeared (, Table S1, and not depicted), whereas at the high doses both old and adult animals succumbed to infection, suggesting that the viral dose is one of the principal determinants of selective mortality of old mice within a specific dose window.
Figure 1. Age-related susceptibility to WNV disease in old mice. (A) Survival of adult (4–6 mo old) and old (18–22 mo old) mice after challenge with the indicated doses of WNV 385–99 s.c. or i.p. Old mice are five and six times more susceptible (more ...)
Figure 2. Relative roles of innate and adaptive immunity and the importance of the age of T cells in resistance to WNV infection. (A) IFNAR−/−, RAG1−/−, and WT mice were infected with the indicated WNV doses s.c., and MST and percentage (more ...)
Figure 5. Inability of aged CD4 and CD8 T cells to protect RAG-1−/− mice against lethal WNV infection. (A) Purified CD8 and CD4 T cells from C57BL/6 (closed squares), IFNγ−/− (open squares), or perforin−/− (more ...)
In principle, this age-related susceptibility could be the result of a generalized inability of old mice to control the virus at the level of both the innate and adaptive immune systems, of the generalized inability to control the virus by one of these two components, or of their focused inability to control the virus in selected target organs. To investigate this issue, infectious viral titer was determined in different organs of adult and old mice and at different time points after infection. After infection with a viral dose that kills most old but not adult mice, we found no significant difference in viral titers between adult and old mice between days 2 and 5 ( and Fig. S2
). Likewise, early time points showed no difference in spleen viral titers (Fig. S2), and our survey of other organs (kidney, gut and liver) also failed to show viral titer differences between old and adult animals between days 3 and 10. We could not detect WNV in the blood past day 5 or in any other organs past day 10, and certain organs (lung and skin, except the site of injection) were negative throughout the course of infection, with the exception of the central nervous system. Indeed, we found significantly higher viral titers in the brains of old mice compared with the adult counterparts on days 8 and 10 after infection (actually between days 7 and 12 [not depicted]; , left and middle). Two additional lines of evidence showed that viral titers within the brain directly correlated to mortality. First, once the animals became moribund, they exhibited equivalent and high brain WNV titers regardless of age (, right). Moreover, when higher infection doses of WNV, lethal to both adult and old mice, were used, both adult and old mice again exhibited comparable and high brain viral titers (unpublished data). Therefore, in all subsequent experiments where we sought to dissect the immunological basis of vulnerability to WNV, we used the viral doses at which most old animals had high viral titers in brain and died but at which the majority of adult mice exhibited low brain WNV titers and survived.
Relative kinetics and roles of innate and adaptive immune responses in mediating defense against WNV infection
We next asked whether the differences mentioned in the previous section could be explained by age-related defects in innate or adaptive immunity. As a model of pronounced innate immunity defect, we used IFN-αβ receptor (IFNAR)–deficient mice (IFNAR−/−
; Müller et al., 1994
), which are unable to respond to type I IFNs (IFN-I) and are known to be highly susceptible to numerous infections (for review see Pestka et al., 2004
), including WNV (Samuel and Diamond, 2005
mice (Mombaerts et al., 1992
), which have no T and B cells as a result of the lack of the recombinase essential for generation of T and B cell receptors and which are also susceptible to many infections, including WNV (Engle and Diamond, 2003
), were used as a model of profoundly deficient adaptive immunity. Our results confirmed that both of these strains are highly susceptible to WNV () but highlighted a difference in the mean survival times (MSTs; ). Thus, IFNAR−/−
mice died rapidly after infection (MST, 5 d), which is consistent with the lack of innate defensive mechanisms, whereas the RAG1−/−
mice died within the same temporal window as old and adult mice (MST, 13 d). Because the MST of old mice was 13–14 d, we concluded that innate immunity in these mice, unlike that in IFNAR−/−
animals, was capable of containing the virus and fending off early WNV-mediated mortality. Therefore, any putative defects in innate immunity in old mice, if present, do not lead to early loss of viral control. To confirm this, we examined functional levels of IFN-I in the serum of adult and old animals. Although the results of this test cannot be taken as fully conclusive because of the low sensitivity of this assay (Fig. S3
), we detected no major age-related differences within these confines. More importantly, any defect in innate immunity would have been expected to result in loss of viral control early after infection. However, this was not observed in any of the experiments designed to test early viremia and viral spread ( and Fig. S2).
Because the results suggested a key role for defects in adaptive immunity, we directly explored this possibility by performing adoptive transfers of old and adult spleen cells (, left) or T cells (CD4 and CD8; , right) into adult RAG1−/− mice. In this series of experiments, we found that adult spleen cells, as well as the adult T cells, conferred significant protection to RAG1−/− mice. In contrast, mice that received old cells were afforded weaker protection, which was not improved in the case of splenocytes and was significant for transferred old T cells (P < 0.04) compared with RAG1−/− mice receiving no cell transfer. Direct comparison between the protective capacity of adult and old T cells revealed significantly better protection by adult T cells (P < 0.003; ). Because antigen presentation and priming are not affected by either age or by the targeted deletion of Rag-1 in the adult RAG1−/− mice, these results collectively strongly suggested that the process of aging impairs resistance to WNV at the level of generation of primary T cell responses.
Age-related differences in WNV-specific T cell responses
IgM and the virus-specific CD8 and CD4 T cells were all implicated in affording protection against WNV in adult mice (Diamond et al., 2003
; Shrestha and Diamond, 2004
; Sitati and Diamond, 2006
). Given the extensive literature on the decline of T cell immunity with aging and our results with T cell transfers, we initiated experiments to test possible defects in the T cell pool. We first examined signs of T cell activation using multicolor flow cytofluorometric analysis. We followed quantitative and qualitative aspects of antigen-specific responses using peptide MHC class I tetramers, as well as the functional response measured by the ability to produce IFN-γ, TNF, and granzyme B (GzB) and express CD43, a molecule involved in T cell activation, costimulation, and effector function (Onami et al., 2002
), and to perform lytic function in response to recently identified immunodominant WNV peptide epitopes that stimulate CD8+
(Brien et al., 2007
) and CD4+
T cells (Brien et al., 2008
; and ). At the peak of the response (days 7–8 after infection), we detected a strongly significant reduction in both percentages and numbers of CD8+
cells specific for the immunodominant class I–restricted epitope NS4b2488
by both tetramer staining () and IFN-γ production after brief in vitro stimulation (). In addition to these quantitative defects, the ratio between IFN-γ–producing and NS4b2488
cells was also significantly reduced in old CD8 cells so that less than one-half of Ag-specific cells were also making the cytokine (). That suggested the existence of superimposed qualitative defects in immunity of old mice against WNV.
Figure 3. Functional quantitative defects in T cell activation in response to WNV infection. Infection was as in . (A) CD8+ T cells derived from spleens of old and adult mice were harvested on day 8 and analyzed for the proportion (left and middle left, (more ...)
Figure 4. Quality of WNV-specific responses in old mice is impaired at several levels. For all panels, animals were infected with 1,000 PFU WNV 385–99 and were analyzed on day 8, unless otherwise indicated. (A) At the peak of infection, splenic CD8 cells (more ...)
To analyze the quality of the anti-WNV response in depth, we examined several aspects of the response, including the ability to produce polyfunctional cells, the intensity of cytokine expression/cell, the level of activation of CD8 cells as measured by CD43 expression, and the ability to lyse peptide-coated cells (). By each and every measure, old CD8 T cells were significantly inferior to the adult counterparts. Thus, although nearly 40% of adult CD8 cells expressed IFN-γ, TNF, and GzB (, 3 Fxn), only 21% of adult cells did so. Second, expression of each of these molecules per cell was significantly lower in old CD8 T cells (). Third, only about half of the WNV-specific (Tet+) CD8 T cells from old mice expressed high levels of CD43, an important activation and function marker (). Finally, direct ex vivo cytotoxicity correlated to GzB expression and was essentially nonexistent in CD8 T cells from old mice compared with adult counterparts ().
These findings were consistent in every experiment performed. Sometimes not all the parameters would reach statistical significance when smaller animal groups were used, but at least three out of four observations were always significant. Similarly, these trends and significant differences were confirmed with the CD8 epitope ENV347
and also in CD4+
cells using the immunodominant CD4+
T cell epitopes (ENV641
) by measuring frequency and magnitude of the IFN-γ (Fig. S4
) and other responses (not depicted). Based on the prior descriptions of signaling defects in CD4 T cells (Garcia and Miller, 2003
; for review see Miller, 1996
), we expected that some of the stated problems in old T cells may trace to inferior ability of old T cells to process antigenic stimulus. However, experiments examining peptide sensitivity of WNV-specific CD8 and CD4 T cells showed superimposable sensitivity of old and adult cells at the peak of the response (Fig. S5
). We conclude that old mice exhibit profound quantitative and qualitative defects in mobilizing fully developed effector T cells but that these defects do not extend to all aspects of antigen recognition and activation.
Mechanisms of T cell–mediated protection against WNV disease and their failure in old mice
We next wanted to determine whether the observed reduction in IFN-γ secretion and/or GzB expression were relevant for protection against WNV in vivo. To that effect, we performed two types of adoptive transfer experiments using RAG1−/−
mice as recipients. First, we transferred total T cells from WT, IFN-γ−/−
, or perforin−/−
donors into RAG1−/−
recipients, which showed that T cells defective in IFN-γ or perforin provide negligible, if any, protection as compared with animals which received no cells at all (). This confirms and extends prior results on the importance of these molecules in anti-WNV protection (Engle and Diamond, 2003
; Wang et al., 2003
) and stresses the critical role of their expression in T cells. However, because old T cells have a reduction but not a complete absence of these molecules, we tested their antiviral ability in vivo by separately transferring highly purified (< 0.5% cross-contamination) CD4 and CD8 T cells from old or adult naive donors into adult RAG1−/−
recipients. These results convincingly showed that either adult CD4 or adult CD8 T cells were sufficient to confer significant protection to RAG1−/−
mice against primary WNV infection in the absence of other components of the adaptive immune system (). More importantly, neither the CD4+
nor the CD8+
T cells from the old mice were able to confer any protection upon RAG1−/−
mice over the level seen in the absence of transfer (), although the combination of the two old T cell subsets did show some synergy, affording a low level of protection (, bottom).
Too little and too weak: insufficient numbers of ineffective effector T cells accumulate in the brains of WNV-infected old mice
If our conclusions were correct, we would expect that they would hold at the level of key organs targeted by WNV. Alternatively, it was also possible that there may be defects in migration and localization of virus-specific T cells to the old brain. To address this issue, we examined the localization and composition of leukocyte infiltrate in the brains of WNV-infected old and adult mice. It is of interest that immunohistochemical examination revealed that in old mice, like in the adult animals, CD3+ cells readily infiltrated the brain parenchyma on days 7 (not depicted) and 12 (), suggesting that there was no major difference in migration and homing. In contrast, the composition of the infiltrate was both qualitatively and quantitatively different (). The large (activated) mononuclear cell gate () contained nearly 3× fewer cells in old mice compared with adult counterparts (3.1 vs. 9.1%, P < 0.0005). Moreover, within that gate, the percentage of CD4 and CD8 cells was also about twofold lower with old age (5.6 vs. 11.4%, P < 0.013, and 14.1 vs. 24.3%, P < 0.037 for CD4 and CD8 cells, respectively; ), leading to a >5× reduction in total CD8 and CD4 cells in old WNV-infected brains. Among these already diminished numbers, the content and quality of virus-specific cells was further reduced. Therefore, the percentage of NS4b-specific tetramer+ CD8 cells specific for WNV was reduced by another twofold (18.6 vs. 38.8%, P < 0.0012; ), with another reduction in GzB-producing cells (trend but not significant at 3.3 vs. 6.7%, P < 0.06; not depicted). Most importantly, when all the reductions were taken into account, compared with the levels found in adult mice (taken as 100%), our analysis suggests that old brains contain at least 12.5× fewer and up to 20× fewer Tet+ GzB+ CD8 T cells () compared with adult brains at the peak of infection.
Figure 6. Analysis of brain infiltrates from WNV-infected adult and old mice. The brains of old and adult C57BL/6 mice were harvested 12 d after infection with 1,000 PFU WNV 385–99 s.c., sectioned, and costained for CD3 (green) and WNV (red). (A) Representative (more ...)
Two important conclusions can be drawn from these results. First, migration to the site of major virus-induced tissue damage is not drastically affected in T cells of old animals. Second, although old T cells apparently arrived to the site of infection, they were not able to differentiate into effector T cells either systemically or locally in numbers sufficient to ensure control of neurovirulence. Therefore, our results identify defects in generation of sufficient number and quality of effector antiviral T cells as the key phenomenon underlying age-related susceptibility to WNV in this model, providing targets for potential therapeutic manipulation.