Cytomegalovirus infection impairs immune responses and accentuates T-cell pool changes observed in mice with aging.

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

Immune Protection against Virus Challenge in Aging Mice Is Not Affected by Latent Herpesviral Infections.

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

Poor CD8 response to WNV in latently infected mice is not caused by viral immune evasive genes.

<p>(A) Mice were primed with 10⁵ PFU of MCMV, 5×10⁵ PFU of ΔMCMV or 10⁶ PFU of VACV-IE1 at 6–8 months of age and challenged with 50 PFU of WNV at 22 months of age. Peptide stimulation with WNV peptides was performed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002849#ppat-1002849-g006" target="_blank">figure 6A</a>, and group averages+SD of IFNγ responses are indicated by histograms. Statistical comparison was performed by ANOVA followed by Bonferroni analysis of individual groups (*** p<0.0001, n.s. p>0.05). (B, C) IFNγ responses upon WNV challenge were correlated to the frequency of (B) EM (CD62L⁻) or (C) naïve (CD11a⁻CD44⁻) CD8 T-cells in individual MCMV (red dots) VACV-IE1 (green dots) or MOCK (black dots) infected mice. Trend indicates the linear correlation, Pearson r and significance (p) are indicated, where the p value indicates the probability that the trend deviates from a horizontal line.</p

Vβ family analysis of CD8 pools upon MCMV infection.

<p>(A,B,C) BALB/cxDBA/2 F1 Mice infected at 6 months of age with MCMV or VACV-IE1 as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002849#ppat-1002849-g001" target="_blank">Fig. 1A</a> and 1B were bled at 14 months following infection and analyzed for frequency of Vβ8, Vβ9, Vβ10, Vβ13 and Vβ14 populations. Representative gating for Vβ 8 and Vβ10 is shown (A). Frequencies of CD8 cells belonging to Vβ families were analyzed in individual mice and a representative analysis is shown for Vβ14 (B), where each mouse is displayed by a symbol, and group medians by horizontal lines. The cohort variability of Vβ population frequencies were defined in groups of MCMV or VACV infected mice (C), and are indicated as SD on the x axis. Variance F-tests were performed to identify differences in group variabilities and p values are indicated in the chart.</p

Cytomegalovirus infection irreversibly perturbs the CD8 T-cell pool.

<p>(A, B) 6 months old mice were infected with 10⁵ PFU of MCMV or 10⁶ PFU of VACV-IE1 and compared to untreated controls. Blood leukocytes were stained for CD3, CD4, CD8, CD11a, CD62L and YPHFMPTNL-L(d) tetramers and analyzed by flow cytometry. (A) CD8 T-cells were gated on tetramer⁺ CD11a⁺ gate as indicated in the representative plot on the left and group means (+/− SD) at 7, 14 28, 60, 90 180, 270 and 420 days post infection are connected. (B) The same cells as in panel A were gated on a CD62L⁻CD11a⁺ gate to identify EM cells (plot on the left) and group means (+/− SD) for indicated days are connected. (C) At 9 months post infection, we gated CD8⁺CD4⁻ lymphocytes and compared the surface TCR expression, defined as the mean fluorescence index (MFI) of CD3, in the MCMV, VACV and mock infected mice. Symbols indicate individual mice, horizontal lines are medians. n.s. – p>0.05; *** - p<0.001 according to Bonferroni statistical comparison. (D) At 9 months post infection, the frequency of activated (Ly6C⁺) cells in the CD8 EM lymphocytes of MCMV, VACV or mock infected mice was compared by Bonferroni statistical comparison. Symbols indicate individual mice, horizontal lines are medians. n.s. – p>0.05; *** - p<0.001.</p

Long-term maintenance of effector and decrease of naïve CD8 subsets upon MCMV infection.

<p>Mouse littermates were infected at 6, 12 or 16 months of age with MCMV (red symbols) or VACV-IE1 (green symbols) and compared at 20 months of age (4, 8 or 14 months post infection, as indicated below x axes) to uninfected controls (black symbols). (A) CD8⁺ cells were gated on CD11a⁺CD44⁺, then on a CD62L⁻ gate, and frequencies of CD11a⁺CD44⁺CD62L⁻ cells in the CD8 pool were calculated. Each symbol represents a mouse, horizontal lines indicate medians. (B) CD8⁺ cells from mice shown in panel A were gated on a KLRG1⁺ gate, and their frequency in individual mice is shown. Each symbol represents a mouse, horizontal lines indicate medians. Significance was assessed by ANOVA followed by Bonferroni post-analysis for indicated columns (* - p<0.05, *** - p<0.001). (C) Naïve cells were defined by progressive gating on a CD11a⁻CD44⁻, and then on a CD127⁺CD62L⁺ gate (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002849#ppat.1002849.s002" target="_blank">Fig. S2</a>). Each symbol represents a mouse, horizontal lines indicate medians. Significance was assessed by ANOVA followed by Bonferroni post-analysis for indicated columns (ns – p>0.05, * - p<0.05, ** - p<0.01, *** - p<0.001).</p

Relative and absolute counts of naïve, CM and EM cells in blood, spleen and LN of MCMV, VACV or MOCK-infected mice.

<p>3 month-old BALB/c mice were injected with 2×10⁵ PFU of MCMV, 10⁶ PFU of VACV or 200 µl PBS (mock infected). 6 months later blood, spleen and inguinal LN CD8⁺ cells were gated on CD11a, CD44, CD62L and CD27 (for a representative gating strategy, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002849#ppat.1002849.s003" target="_blank">Fig. S3</a>) to define (A) the relative representation and (B) the absolute count of naïve, CM and EM subsets in each compartment. Each dot represents data from a single mouse, horizontal lines show medians.</p

Murine cytomegalovirus infection via the intranasal route offers a robust model of immunity upon mucosal CMV infection.

Cytomegalovirus (CMV) is a ubiquitous virus, causing the most common congenital infection in humans, yet a vaccine against this virus is not available. The experimental study of immunity against CMV in animal models of infection, such as the infection of mice with the mouse CMV (MCMV), has relied on systemic intraperitoneal infection protocols, although the infection naturally transmits by mucosal routes via body fluids containing CMV. To characterize the biology of infections by mucosal routes, we have compared the kinetics of virus replication, the latent viral load, and CD8 T cell responses in lymphoid organs upon experimental intranasal and intragastric infection to intraperitoneal infection of two unrelated mouse strains. We have observed that intranasal infection induces robust and persistent virus replication in lungs and salivary glands, but a poor one in the spleen. CD8 T cell responses were somewhat weaker than upon intraperitoneal infection, but showed similar kinetic profiles and phenotypes of antigen-specific cells. On the other hand, intragastric infection resulted in abortive or poor virus replication in all tested organs, and poor T cell responses to the virus, especially at late times after infection. Consistent with the T cell kinetics, the MCMV latent load was high in the lungs, but low in the spleen of intranasally infected mice and lowest in all tested organs upon intragastric infection. In conclusion, we show here that intranasal, but not intragastric infection of mice with MCMV represents a robust model to study short and long-term biology of CMV infection by a mucosal route

CD8 T-cell responses to superinfection upon MCMV infection.

<p>(A) groups of 3 (young) and 12 (old) month old mice were primed with 10⁵ PFU of MCMV, 10⁶ PFU of VACV-WR or PBS (MOCK), and challenged 5 months later with 300 EID of influenza virus, PR/8 strain, intranasally (i.n.). 7 days post challenge blood lymphocytes were tested by ICCS for IFNγ responses to a 9 h in vitro stimulation with the NP³⁶⁶ peptide in the presence of BrefeldinA. Mean IFNγ responses in animal groups (n = 5/group)+SD is shown on the y axis. (B) 12 month-old mice were primed as indicated and challenged 5 months later with Influenza. The frequency of peptide specific cells in the blood CD8 pool was defined by peptide restimulation and ICCS. Group average (+SD) values for either peptide are indicated on the y axis. (C) Mice were primed with 10⁵ PFU of MCMV or 10⁶ PFU of VACV-IE1 at 2 months of age, and i.p. challenged with 50 PFU of West Nile Virus at 8 months of age. 7 days later, blood lymphocytes were in vitro stimulated with a pool of two immunodominant H^2d restricted WNV peptides and analyzed by ICCS. The frequency of IFNγ expressing T-cells in the CD8 pool of individual mice is displayed on the y axis. Horizontal lines indicate medians. (D) C57BL6/DBA2 F1 mice were primed with MCMV or VACV-IE1 at 3 months of age, challenged with HSV-1 at 8 months of age and frequencies of HSV-1 specific responses were determined 7 months later by pMHC tetramer staining and FCM. Each mouse is indicated with a symbol, horizontal lines are means, the dashed line shows the detection threshold. The p value for Mann-Whitney analysis is shown. (E, F) Mice were primed with MCMV or VACV-IE1 at 6, 12, 16 or 20 months of age, and assayed for responses to WNV challenge at 22 months of age (16, 10, 6 or 2 months post prime, as indicated below axis) using the protocol as in panel C. Each mouse is indicated with a symbol, horizontal lines are means. (E) % of CD8 cells responding to peptide stimulation in ICCS. (F) Leukocytes were in parallel assayed by polyclonal stimulation with anti-CD3 antibodies, and the peptide specific response was normalized to the CD3 (Max) response. Values in panels A, C, E and F were compared by 1-way ANOVA followed by Bonferroni comparison of individual columns, and significance is indicated (n.s. - p>0.05, *- p<0.05, ** - p<0.01, *** - p<0.001).</p