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. 2012;8(8):e1002849.
doi: 10.1371/journal.ppat.1002849. Epub 2012 Aug 16.

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

Luka Cicin-Sain  1 , James D Brien, Jennifer L Uhrlaub, Anja Drabig, Thomas F Marandu, Janko Nikolich-Zugich
Affiliations

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

Luka Cicin-Sain et al. PLoS Pathog. 2012.

Abstract

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Cytomegalovirus infection irreversibly perturbs the T-cell pool.
Blood leukocytes of mice infected with 105 PFU of MCMV, 106 PFU of VACV-IE1 or injected with PBS were analyzed at 9 months post infection by flow cytometry and representative gating is shown (A). Pecentages of CD4+ T-cells (B) or CD8+ T-cells (C) in the lymphocyte pool were divided in individual mice to calculate the CD4/CD8 ratio (D). Cells were in parallel acquired in a cell counter, and the lymphocyte count was multiplied with the fraction of CD4 or CD8 cells in the lymphocyte population, to calculate the absolute number of CD4 T-cells (E) or CD8 T-cells (F) per ml of blood. The percentage of cells out of either gate was multiplied with the lymphocyte count to establish the absolute count of non-T lymphocytes (G). Symbols denote values from individual mice, horizontal lines show medians. Significant (p<0.05) group differences upon ANOVA and Dunnet Post analysis are indicated, where * - p<0.05; ** - p<0.01; *** - p<0.001.
Figure 2
Figure 2. Cytomegalovirus infection irreversibly perturbs the CD8 T-cell pool.
(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.
Figure 3
Figure 3. Long-term maintenance of effector and decrease of naïve CD8 subsets upon MCMV infection.
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 Fig. S2). 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).
Figure 4
Figure 4. Vβ family analysis of CD8 pools upon MCMV infection.
(A,B,C) BALB/cxDBA/2 F1 Mice infected at 6 months of age with MCMV or VACV-IE1 as in Fig. 1A 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.
Figure 5
Figure 5. Relative and absolute counts of naïve, CM and EM cells in blood, spleen and LN of MCMV, VACV or MOCK-infected mice.
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 Fig. S3) 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.
Figure 6
Figure 6. CD8 T-cell responses to superinfection upon MCMV infection.
(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 H2d 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).
Figure 7
Figure 7. Poor CD8 response to WNV in latently infected mice is not caused by viral immune evasive genes.
(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 figure 6A, 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.
Figure 8
Figure 8. Poor mobilization of CD8 cells in draining LN of MCMV infected mice upon flu challenge.
LN from 9 month old BALB/c mice infected with VACV or MCMV for six months prior to intranasal challenge with influenza virus were collected at dpi 7. (A) Cells were gated on the CD8+ CD4− gate in an Accuri cytometer to establish absolute counts of CD8+ cells per mesenteric (Mes-LN), inguinal (Ing-LN) or mediastinal (Med-LN) lymphnodes. (B) CD8 cells were acquired in parallel in an LSR2 apparatus and analyzed as shown in supplementary figure 3. Dividing the CD8 cell count with the fraction of cells in the EM gate defined the absolute counts for this subset. Cell counts in individual mice are displayed, horizontal lines indicate medians. 1-way Anova followed by Bonferroni post analysis was performed to define the differences between draining and non-draining LN (***-p<0.001, **-p<0.01, *-p<0.05, n.s.-p>0.05).
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