In this study, we demonstrate that experimental CMV infection diminishes the CD8 response to heterologous virus infections. It has been shown that herpesvirus infections have the potential to improve the immune protection against bacterial infections 
due to elevated innate immune responses. In our study we observed no positive effects of latent MCMV infection on the immune protection of mice upon WNV challenge, if anything the survival was slightly shorter and lower (L.C-S. & J.N-Z., unpublished data). During the preparation of this manuscript, we became aware of the results by the Karrer group (Mekker et al, Submitted as companion manuscript), whose results further support this interpretation – these authors found that mice latently infected with MCMV show diminished clearance of lymphoid choriomeningitis virus (LCMV) upon challenge (Mekker et al. submitted). The differences between our data and those of Barton et al 
likely reflect differences in challenge models and perhaps more importantly, the age of experimental animals and/or the length of CMV infection and the time of testing after primary infection. While that study focused on effects occurring relatively soon after infection (45 days post infection) in young adult mice, we focused on effects occurring 5 months or later upon infection, in middle aged or old mice.
We considered the possibility that the absolute number of CD8 cells responding to WNV or flu remained unchanged, and that the lower frequencies of responding cells merely mirrored an absolute increase of memory cells due to the CMV-induced T-cell expansions. However, while CMV infection increased the size of the peripheral blood CD8 pool by a factor of 1.7 (), this could not have explained the observed 10 or 12-fold differences in the frequency of flu-specific cells (see ), which was consistent with the absolute loss of CD8 response to LCMV challenge observed in old MCMV-infected mice (Mekker et al. submitted).
The phenotype analysis of the blood compartment argued for two main possibilities: (i) that a loss of naïve T cell numbers, and/or their TCR repertoire diversity, caused poor responses to superinfection with emerging viruses (this scenario could explain the difference between MCMV and mock-infected mice based on numerical differences, but not between MCMV and VACV infected mice, since both infections reduced the naïve CD8 count in the blood; potential TCR repertoire changes remain to be analyzed in sufficient detail); or (ii) that accumulation of large EM cell populations inhibited the response of the remaining naïve cells, which was in line with findings of others (Mekker et al. submitted). The analysis of compartments other than the blood showed an overall accumulation of CD8 cells with no loss of the naïve subset, excluding the reduced naïve cell numbers as the mechanism underlying poor responses in MCMV infected mice. Moreover, in LN of MCMV infected mice we observed a specific enrichment of the CM and not the EM fraction. This is in line with data by Snyder et al. showing that the MCMV-specific EM fraction of memory-inflated cells cannot renew on its own, but rather mirrors the proliferation of a CMV-specific subset of CM cells in the LN of infected mice 
. Finally, and most importantly, upon an influenza challenge we observed an overall expansion of the CD8 pool in the draining LN, which was impaired in MCMV-infected mice. Detailed analysis showed that this impairment was mainly due to poor expansion and/or accumulation of effector CD8 cells. Therefore, while our results do not formally exclude the possibility that the poor immune responses were caused by other mechanisms, they suggest that latent MCMV infection specifically contributes to the poor activation of CD8 cells in the LN of mice carrying latent MCMV.
Mekker et al. also observed that young animals infected for two months with MCMV prior to challenge showed less reduction in CD8 responses to LCMV challenge than old animals, while we observed an opposite trend in adult and old mice challenged with influenza at 5 months post CMV infection. This difference likely reflects the differences in age at challenge and length of MCMV infection prior to infection, or in the choice of viruses used for challenge. Interestingly, when we normalized the age of mice at challenge, but infected mice with MCMV at different ages, we observed that the CD8 response to superinfection with WNV was diminished in MCMV infected animals, but was not worsening with the passage of time upon MCMV infection (), Similarly, VACV infection resulted in a transient increase of EM and of KLRG1+ fractions, and the only groups of MCMV and VACV infected animals that showed no significant difference to one another in responses to WNV challenge were the groups of animals infected for two months prior to challenge (). Nothwithstanding these minor discrepancies, our results described above and the data from Mekker et al. are highly consistent and demonstrate that MCMV infection results in several phenotypical and functional changes of the immune system that are usually associated with aging.
Surprisingly, we observed that MI does not reflect a progressive expansion of the entire EM pool. In fact, after its initial rapid expansion, the fraction of EM cells in the CD8 pool did not increase further than the levels seen 14 days after infection (). On the other hand, the CD8 cells recognizing the immunodominant epitope YPHFMPTNL increased in the same mice from approximately 2 to 16% of the CD8 compartment. This implies that after the initial expansion of EM cells, the main change within the CD8 compartment did not involve their further expansion, but rather the replacement of the relatively polyclonal antiviral response by the oligoclonal response against defined epitopes, while the overall magnitude of the response remained unaltered. Therefore, our results raise the question whether the increase of CMV pp65 Ag specific cells observed with advancing age 
, indeed reflects an increase in the population of CMV specific cells, or merely reflects focusing of CMV-specific CD8 responses upon a narrower range of defined immunodominant targets.
Numerous clinical studies showed that CMV seropositivity coincides with poor responses to other viruses 
, poor responses to vaccines 
, or poor life expectancy in the very old 
. We showed recently that the phenotype and function of CMV-specific T-cells from old and immunosenescent rhesus monkeys are indistinguishable from those in adult and immunocompetent ones 
. However, the rhesus model did not allow the comparison of CMV infected and uninfected hosts, because essentially all captive rhesus monkeys become naturally infected with CMV early in their life. Therefore, the effects of CMV infection on the aging immune system could only be defined in mouse models of infection, where SPF colonies allow the maintenance of MCMV negative controls throughout their lifetime. To our knowledge, along with the study by Mekker et al., this was the first attempt to establish an experimental model to study this question and the first experimental proof that a persistent herpesvirus infection may lead to an irreversible change in the CD8 pool and impair their ability to respond to emerging infections. While Mekker et al showed clearly that MCMV infection results in a loss of relative and absolute responses to superinfection with unrelated viruses, we showed that the poor responses were exclusive to infection with MCMV and could not be observed in infections with other viruses, such as vaccinia or Herpes simplex, and showed that poor responses are observed in several murine strains and F1 hybrids, arguing that the phenomenon is independent of the mouse gentoype. Mekker et al. showed by adoptive transfer experiments that MCMV infection does not compromise the ability of donor cells to respond to an antigen that they recognize, arguing that MCMV did not impair CD8 function in general, but only the endogenous responses to viruses, whereas we showed that none among the known MCMV immune evasive genes contributes to the suppression of CD8 responses to challenge. Instead we could show a decrease in the recruitment and/or activation of CD8 cells in draining LN upon a viral challenge. Therefore, our results and those from Mekker et al. may have profound implications for our understanding of the immune aging process in a microbiological environment that reflects the real-life exposure of the average individual.
While we focused in this study on the CD8 subset, our data do not exclude the possibility that latent MCMV infection may affect the CD4 T-cell and the B-cell subsets of lymphocytes. Future studies will allow us to elucidate the effect of persisting CMV infections on T-helper subsets and on humoral immune responses, a question particularly important in light of the relevance of these subsets for the efficiency of vaccination strategies.
Based on our data, we propose a model in which the persistent CMV infection causes an ongoing recruitment of EM cells in in lymph nodes, which impairs the ability of the naïve cells to mount responses to unrelated virus. Future studies should elucidate whether and to what extent these effects are due to competition of T-cell populations within the lymph node, or alterations in the migration and/or antigen presenting function of professional APC, or whether there also may be a component of TCR repertoire diversity reduction, for which there is some support (Smithey, M.J. et al., submitted for publication). The newly established balance may offer benefits in terms of stronger innate immune responses 
, yet at the same time diminishes the efficiency of the adaptive branch to target neoantigens. All of these changes, combined with age-related cell-autonomous changes in T-cells 
or other parts of the immune system (rev in 
) could contribute to the clinical evidence of poor prognosis and poor CD8 function in very old CMV-positive hosts observed in some studies 
. Our model should provide a useful tool to elucidate the effects of persistent CMV infection on the immune system and the mechanisms of this interaction.