Einfluss von Anhaltender Antigenexpression auf Zytomegalievirus-spezifische T-Zellantworten

More than 90% of the human population is latently infected with Cytomegalovirus (CMV). Infectious virus is not produced during latency, but viral genomes remain in infected host cells for years and retain the ability to reactivate upon episodes of immune suppression. The murine CMV (MCMV) is one of the most commonly used in vivo models of CMV infections. During viral latency, the immune system is weakly, but constantly, stimulated by viral antigens, resulting in an expansion of CMV-specific CD8+ T cells with an effector memory phenotype, a phenomenon termed Memory Inflation (MI). It is assumed that these cells are short-lived, but regularly replenished with antigen specific cells from other T cell subsets. To define whether expression of the ie1 and ie3 genes during latency is responsible for maintenance of MI, the recombinant virus MCMV IE1/3flox was generated by flanking the ie1/3 transcriptional unit with loxP sites. Mice expressing a Tamoxifen (Tam)-inducible Cre recombinase (Cre.ERT2) were infected with MCMV IE1/3flox or MCMV WT. The administration of Tam at 4 months post infection allowed the selective targeting of loxP sites at times of viral latency. The percentage of CMV-specific inflationary CD8+ T cells and the relative size of the effector memory T (TEM) cell pool were transiently decreased upon Tam administration, in mice infected with MCMV IE1/3flox, but also in those infected with MCMV WT. This contradiction could be explained by direct toxicity of Cre.ERT2 on proliferating T cells. Analysis of the CD8+ T cell subsets in the spleens of latently infected Cre.ERT2 mice revealed increased apoptosis in TEM cells only. This suggests that the observed reduction of TEM cells was not caused by the elimination of cycling naive or central memory T cells and consequently less recruitment of these cells to the TEM cells. In conclusion, the results argue that inflationary CD8+ T cells and TEM cells in general are maintained, at least in part, by cyclic TEM cells.Mehr als 90% der menschlichen Bevölkerung sind latent mit dem Zytomegalievirus (CMV) infiziert. In der Latenz werden keine Viren produziert, aber virale Genome lassen sich in infizierten Wirtszellen nachweisen. In Folge von Immunsuppressionen kann das Virus reaktivieren. Das murine CMV (MCMV) ist eines der meistgenutzten in vivo Modelle der CMV-Infektion. Während der viralen Latenz wird das Immunsystem zwar nur gering, aber permanent durch virale Antigene stimuliert, wodurch es zu einer Expansion von CMV- spezifischen Effektorgedächtniszellen (TEM) kommt, ein Phänomen bezeichnet als „Memory Inflation“ (MI). Um zu klären, ob die Expression der Gene ie1 und ie3 während der Latenz für die MI verantwortlich ist, wurde das rekombinante Virus MCMV IE1/3flox erschaffen, bei dem die Transkriptionseinheit von ie1/3 mit loxP-Stellen flankiert wurde. Mäuse, welche eine Tamoxifen (Tam)-induzierbare Cre-Rekombinase (Cre.ERT2) exprimieren, wurden mit MCMV IE1/3flox oder MCMV WT infiziert. Cre.ERT2 wurde 4 Monate nach der Infektion durch die Verabreichung von Tam aktiviert. Der Anteil der inflationären MCMV-spezifischen CD8+ T-Zellen und der TEM-Zellen waren nach der Verabreichung von Tam in MCMV IE1/3flox-infizierten, aber auch in MCMV WT-infizierten Mäusen vorübergehend reduziert. Dieser Widerspruch könnte durch eine direkte Toxizität der Cre.ERT2 auf proliferierende T- Zellen erklärt werden. Die Analyse verschiedener CD8+ T-Zellsubpopulationen aus der Milz latent infizierter Cre.ERT2 Mäuse, ergab einen höheren Anteil apoptotischer Zellen ausschließlich in der TEM-Zellsubpopulation. Dies deutet darauf hin, dass die Reduzierung von TEM-Zellen nicht durch die Eliminierung proliferierender naiver T-Zellen oder TCM-Zellen verursacht wurde, was zu weniger Rekrutierung dieser Zellen zur TEM-Zellsubpopulation führen würde. Dies könnte bedeuten, dass die Anzahl von inflationären CD8+ T-Zellen und TEM-Zellen, zumindest teilweise, durch zyklische TEM-Zellen aufrechterhalten bleibt

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

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

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

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

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

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

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