Prominent immune alterations associated with aging include the loss of naïve T-cell numbers, diversity and function. While genetic contributors and mechanistic details in the aging process have been addressed in multiple studies, the role of environmental agents in immune aging remains incompletely understood. From the standpoint of environmental infectious agents, latent cytomegalovirus (CMV) infection has been associated with an immune risk profile in the elderly humans, yet the cause-effect relationship of this association remains unclear. Here we present direct experimental evidence that mouse CMV (MCMV) infection results in select T-cell subset changes associated with immune aging, namely the increase of relative and absolute counts of CD8 T-cells in the blood, with a decreased representation of the naïve and the increased representation of the effector memory blood CD8 T-cells. Moreover, MCMV infection resulted in significantly weaker CD8 responses to superinfection with Influenza, Human Herpes Virus I or West-Nile-Virus, even 16 months following MCMV infection. These irreversible losses in T-cell function could not be observed in uninfected or in vaccinia virus-infected controls and were not due to the immune-evasive action of MCMV genes. Rather, the CD8 activation in draining lymph nodes upon viral challenge was decreased in MCMV infected mice and the immune response correlated directly to the frequency of the naïve and inversely to that of the effector cells in the blood CD8 pool. Therefore, latent MCMV infection resulted in pronounced changes of the T-cell compartment consistent with impaired naïve T-cell function.

Herpesviruses establish a lifelong latent infection posing the risk for virus reactivation and disease. In cytomegalovirus infection, expression of the major immediate early (IE) genes is a critical checkpoint, driving the lytic replication cycle upon primary infection or reactivation from latency. While it is known that type I interferon (IFN) limits lytic CMV replication, its role in latency and reactivation has not been explored. In the model of mouse CMV infection, we show here that IFNβ blocks mouse CMV replication at the level of IE transcription in IFN-responding endothelial cells and fibroblasts. The IFN-mediated inhibition of IE genes was entirely reversible, arguing that the IFN-effect may be consistent with viral latency. Importantly, the response to IFNβ is stochastic, and MCMV IE transcription and replication were repressed only in IFN-responsive cells, while the IFN-unresponsive cells remained permissive for lytic MCMV infection. IFN blocked the viral lytic replication cycle by upregulating the nuclear domain 10 (ND10) components, PML, Sp100 and Daxx, and their knockdown by shRNA rescued viral replication in the presence of IFNβ. Finally, IFNβ prevented MCMV reactivation from endothelial cells derived from latently infected mice, validating our results in a biologically relevant setting. Therefore, our data do not only define for the first time the molecular mechanism of IFN-mediated control of CMV infection, but also indicate that the reversible inhibition of the virus lytic cycle by IFNβ is consistent with the establishment of CMV latency.

f Latent herpesvirus infections alter immune homeostasis. To understand if this results in aging-related loss of immune protection against emerging infections, we challenged old mice carrying latent mouse cytomegalovirus (CMV), herpes simplex virus 1 (HSV-1), and/or murine gammaherpesvirus 68 (MHV-68) with influenza virus, West Nile virus (WNV), or vesicular stomatitis virus (VSV). We observed no increase in mortality or weight loss compared to results seen with herpesvirus-negative counterparts and a relative but not absolute reduction in CD8 responses to acute infections. Therefore, the presence of herpesviruses does not appear to increase susceptibility to emerging infections in aging patients.

20Effector-memory T-cells (TEM) are assumed to be short-lived cells that poorly proliferate upon 21 antigenic restimulation, thus depending on central-memory T-cells (TCM) to replenish their numbers 22 during homeostasis, largely depending on adoptive transfer evidence. Here we analyzed T cells in 23 their natural environment and observed robust long-term in vivo cycling within the TEM subset that 24 was stronger than the one in the TCM subset. Murine Cytomegalovirus (MCMV) induces inflationary 25 TEM responses that remain high during latency. We analyzed Ki67 expression during acute and latent 26 MCMV infection and found Ki67 hi Bcl2 lo TEM in latently infected mice, arguing for antigen-driven TEM 27 proliferation. TEM acquired deuterium more rapidly than TCM in an in vivo labeling experiment, and 28 were replenished more rapidly than TCM after memory depletion, suggesting that TEM cycle faster 29 than TCM. We depleted selectively the proliferating T-cells by Cre-overinduction, which resulted in a 30 selective loss of Ki67 hi Bcl2 lo effector T-cells, and an increase in the death of TEM in the spleen, while 31 it hardly affected the TCM subset, arguing for robust proliferation of TEM in the spleen. On the other 32 hand, TEM homing to the spleen upon adoptive transfer was substantially poorer than TCM, 33 explaining the previously reported expansions of TCM, but not TEM, upon transfer. In conclusion, our 34 data suggest that memory inflation is maintained by proliferation of antigen-specific TEM, rather 35 than by continued expansion and differentiation of TCM. 36 37 Running title: TEM proliferation during MCMV latency 3 38 Author Summary 39 The naïve T cell population consists of T cells that have the potential to recognize millions of 40 different pathogens. Upon infection, naïve T cells that recognize the pathogen expand, and 41 differentiate into effector T cells that eliminate infected cells. Once the infection is contained, the T 42 cell pool contracts and only a small population of central memory T cells remains that can expand 43 quickly upon re-infection. Cytomegaloviruses cause persistent infections that are not cleared from 44 the organism after the initial immune response. In infected individuals a pool of CMV-specific 45 effector memory T cells dominates the immune system in a phenomenon called memory inflation. 46 Previous research using the transfer of central memory or effector memory T cells from CMV-47 infected mice into mice with a matching infection, showed expansion of central memory T cells but 48 not effector memory T cells. Here we show that effector memory T cells have a reduced capacity to 49 home into lymphoid organs, where T cell activation takes place, compared to central memory T cells. 50 Using methods that do not interfere with T cell differentiation and homing, we show that effector 51 memory T cells are proliferating during the persistent phase of CMV infection, significantly 52 contributing to the upkeep of the inflationary population. 53 126 Ki67 hi CD8 T-cells was substantiall...

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