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. Author manuscript; available in PMC: 2013 May 16.
Published in final edited form as: J Immunol. 2010 Aug 16;185(6):3583–3592. doi: 10.4049/jimmunol.1001535

Interleukin-10 restricts memory T cell inflation during cytomegalovirus infection

Morgan Jones *, Kristin Ladell *, Katherine K Wynn *, Maria A Stacey *, Máire F Quigley *, Emma Gostick *, David A Price *, Ian R Humphreys *,1
PMCID: PMC3655265  EMSID: EMS53114  PMID: 20713884

Abstract

The beta-herpesvirus cytomegalovirus induces a substantial and progressive expansion of virus-specific memory CD8 T cells, which protect the host against viral reactivation from latency. Here, we report that this expansion, or “inflation”, of memory T cells is amplified dramatically during mouse cytomegalovirus (MCMV) infection of interleukin-10 (IL-10) knockout (IL-10−/−) mice. T cells from IL-10−/− mice were oligoclonal, exhibited a highly activated phenotype, expressed antiviral cytokines and degranulated in response to cognate antigen encounter ex vivo. Moreover, latent viral load was reduced in IL-10−/− mice. Importantly, these results were recapitulated by IL-10 receptor blockade during chronic/latent infection of wild-type mice. These data demonstrate that regulatory immune mechanisms can influence cytomegalovirus-specific T cell memory and suggest a possible rationale for the acquisition of functional IL-10 orthologs by herpesviruses.

Keywords: CD8 T cell, cytomegalovirus, IL-10, memory

Introduction

Human cytomegalovirus (HCMV) is a β-herpesvirus that infects 70-90% of the human population. Although primary HCMV infection is typically asymptomatic, it causes multi-organ disease in unborn children and immune compromised seronegative adults (1). HCMV also establishes a latent infection that is generally well contained by healthy hosts. However, in individuals with impaired immune function, such as transplant recipients receiving immunosuppressive drugs, latent virus can reactivate and induce severe clinical disease (2, 3). Furthermore, indirect effects such as acute and chronic graft rejection can be triggered by these events (3).

CD8 T cells are critical mediators of antiviral immune responses to HCMV. Viral reactivation in immune suppressed individuals is associated with a numerical and functional impairment of virus-specific CD8 T cells (4-7), and adoptive transfer of CD8 T cells affords protection from virus reactivation and viremia (8-11). In addition, using the mouse cytomegalovirus (MCMV) model of infection, it has been shown that adoptive transfer of CD8 T cells protects immune suppressed hosts from infection (12-15). Critically, MCMV reactivation from latency is also controlled by CD8 T cells (16).

CMV-specific CD8 T cells expand to unusually high frequencies in immune competent humans and mice (17, 18), a phenomenon that has been termed “memory inflation” (19). During MCMV infection, the memory CD8 T cell pool consists of “stable” memory T cells that are present at low numbers and “inflationary” memory T cell populations that expand to high frequencies as infection progresses (19-21). Inflation of memory T cells is antigen-dependent (22), and phenotypic analysis suggests that these cells are highly differentiated and have recently been exposed to antigen (20, 21, 23-25); thus, it appears that periodic viral reactivation events drive the expansion of these cells. However, it is unclear whether immunological factors also regulate this process.

Interleukin 10 (IL-10) is an immune regulatory cytokine that suppresses T cell responses primarily via effects mediated on antigen-presenting cells (APC); specifically, IL-10 inhibits the expression of pro-inflammatory cytokines and chemokines, MHC class II and costimulatory molecules by APC (26, 27). During viral infections, IL-10 has paradoxical functions. In models of acute influenza (28) and herpes simplex infection (29), IL-10 limits immunopathology. In contrast, IL-10 antagonizes protective immunity following high dose influenza challenge (30) and MCMV persistence in the salivary gland (31). Moreover, IL-10 induction during chronic lymphocytic choriomeningitis virus (LCMV) infection promotes functional dysregulation of T cells and lymphopenia, thereby enabling prolonged viral replication (32, 33). The expression of a functional IL-10 ortholog (vIL-10) by HCMV indicates the importance of IL-10 in the suppression of HCMV-specific immunity (34, 35). Intriguingly, in vitro experiments have demonstrated vIL-10 gene expression during latent HCMV infection (36) and suggest that vIL-10 may inhibit memory CD4 T cell recognition of latently infected cells (37). To date, however, nothing is known regarding the role of IL-10 in the regulation of cytomegalovirus-specific memory T cell responses in vivo.

In the current study, we show that IL-10 is a potent inhibitor of memory T cell inflation during MCMV infection. We observed a massive accumulation of T cells expressing antiviral cytokines in MCMV-infected IL-10 knockout (IL-10−/−) mice. This was associated with enhanced CD4 T cell immunity and APC recruitment to sites of viral latency. Using an antibody that blocks the IL-10 receptor (IL-10R), we also demonstrated that IL-10 inhibits memory T cells during chronic/latent infection. Critically, enhanced immunity at this time following either IL-10 gene deletion or IL-10R blockade led to a reduction in viral load. Collectively, these data suggest that IL-10 promotes the long-term maintenance of CMV by limiting the expansion of functional antiviral memory T cells.

Material and Methods

Mice and ethics

IL-10−/− mice were purchased from Jackson Laboratory and bred in-house; wild-type C57BL/6 mice were purchased from Harlan UK. All experiments were conducted according to UK Home Office guidelines. Specifically, these experiments were performed under a Home Office project license (granted to I.H.) in the Home Office-designated facility at Heath Park, Cardiff University.

Virus, mouse infections and treatments

MCMV Smith strain (American Type Culture Collection, ATCC) was prepared in BALB/c salivary glands and titered on 3T3 cells as described previously (38). Mice were infected intra-peritoneally (i.p.) with 5 × 104 pfu MCMV. In some experiments, wild-type mice were injected on the days stated in the figure legends with 250 μg of either rat IgG (Chemicon) or αIL-10Rα (clone 1B1.3A, BioXCell).

Intracellular cytokine staining and flow cytometry

To evaluate MCMV-specific CD8 T cell responses, splenocytes and lung leukocytes were isolated from infected mice and incubated at 5 × 106 cells/ml for 5 hours at 37°C with 2 μg/ml MCMV-derived peptides (Genscript) in the presence of either 0.7 μg/ml monensin and αCD107a-FITC (both BD Pharmingen; IFN–γ and CD107a detection) or 2 μg/ml brefeldin A (Sigma-Aldrich; IFN–γ, TNF and IL-2 detection). The optimal length peptides used in this study were: (i) SSPPMFRVP (origin, M38; restriction, H-2Kb); (ii) HGIRNASFI (origin, M45; restriction, H-2Db); (iii) TVYGFCLL (origin, m139; restriction, H-2Kb); and, (iv) RALEYKNL (origin, IE3; restriction, H-2Kb) (20). Cells were then washed and incubated with Fc block (eBioscience). After incubation with peptide or medium alone as a control, cells were surface stained with αCD8α-APC-H7 (BD Pharmingen). All cells were then fixed with 3% formalin, permeabilized with saponin buffer (PBS, 2% FCS, 0.05% sodium azide and 0.5% saponin) and stained with the relevant combinations of αTNF-PE-Cy7, αIL-2-Pacific Blue and αIFN-γ-FITC (all eBioscience). To calculate % virus-specific effector function, spontaneous degranulation or cytokine production in medium controls was subtracted from peptide-induced mobilization or expression, respectively.

To identify MCMV-specific CD4 T cells, splenocytes and lung leukocytes were incubated for 5 -16 hours at 37°C with 2 μg/ml brefeldin A (Sigma-Aldrich) and 3 μg/ml MCMV-derived peptides (Genscript) restricted by MHC class II as follows: (i) GYLYIYPSAGNSFDL (origin, m09); (ii) NHLYETPISATAMVI (origin, M25); (iii) TRPYRYPRVCDASLS (origin, m139); and, (iv) RSRYLTAAAVTAVLQ (origin m142) (39). To identify IL-10 expression by B cells and macrophages, splenocytes were stimulated for 5 hours at 37°C with either 10μg/ml LPS (macrophages) or 10μg/ml LPS, 50ng/ml PMA and 500ng/ml ionomycin (B-cells; all reagents from Sigma-Aldrich). Cells were then incubated with Fc block and surface stained with either αCD4-Pacific Blue (BD Pharmingen), αCD19-FITC or αCD11b-PE-Cy7 prior to permeabilization and intracellular staining with αIFN-γ-FITC (CD4 analysis only) and αIL-10-APC (all eBioscience). The % virus-specific cytokine production by CD4 T cells was calculated as described for CD8 T cells.

To assess APC accumulation, unstimulated splenocytes and lung leukocytes were incubated with Fc block and then stained with αCD11b-APC-Cy7 (BD Pharmingen), αCD11c-PE-Cy7 (BD Pharmingen), αMHC class II-PE-Cy5 (eBioscience), αF4/80-Pacific Blue (BioLegend), αGr1-FITC (BioLegend) and α7/4-PE (Serotec).

To examine the expression of surface molecules by MCMV-specific CD8 T cells, 1 × 106 splenocytes were incubated with Live/Dead Fixable Aqua® (Invitrogen) followed by Fc block, then stained with H-2Kb tetramers loaded with M38, m139 or IE3 peptides. Cells were then stained with αCD3-PerCP-Cy5.5 (BD Pharmingen), αCD8-APC-H7 (BD Pharmingen) and either αCD44-FITC (BioLegend), αCD62L-PE-Cy7 (Abcam) and αCD122-PE (eBioscience), or αKLRG-1-FITC (Southern Biotech), αCD27-PE (eBioscience) and αCD127-Pacific Blue (eBioscience).

All data were acquired on either a BD FACS Canto II or a modified FACS Aria II cell sorter equipped for the detection of 18 fluorescent parameters (BD Immunocytometry Systems). Electronic compensation was performed in all cases using antibody-capture beads stained separately with the individual mAbs used in each experimental panel. A minimum of 30,000 events were acquired in each case and data were analyzed using FlowJo software version 8.5.3 (TreeStar Inc.).

In all experiments, total numbers of different cell populations were calculated by multiplying the total number of viable splenic or pulmonary leukocytes (assessed by trypan blue exclusion) by % positive cells, as detected by flow cytometry. Total numbers of virus-specific T cells were calculated by: total T cell number (CD4 or CD8) × % peptide-specific IFN–γ+ cells (calculated as described above). Total numbers of virus-specific TNF+ or CD107a+ T cells were calculated similarly.

TCR clonotyping

Clonotypic analysis of cognate antigen-specific CD8 T cell populations was conducted as described previously with minor modifications (40, 41). Briefly, tetramer-labelled CD8 T cells (median: 3,200; range: 808-5,000) were sorted viably into 1.5 ml microtubes containing 100 μl RNAlater (Applied Biosystems) and unbiased amplification of all expressed TCRB gene products was conducted using a template-switch anchored RT-PCR with a 3′ TCRB constant region primer (5′-TGGCTCAAACAAGGAGACCT-3′). Amplicons were subcloned, sampled, sequenced and analyzed as described previously (42). The IMGT nomenclature is used throughout this report (43).

Viral genome detection

Genomic DNA was isolated from spleen and lung tissue (Qiagen) and MCMV glycoprotein B (gB) was then assayed by quantitative PCR using a Mini Opticon (Biorad Laboratories) and Platinum SYBR green mastermix reagent (Invitrogen). 100 ng aliquots of DNA were used as templates for each reaction. The primer sequences used for detection of β-actin were 5′-GATGTCACGCACGATTTCC-3′ and 5′-GGGCTATGCTCTCCCTCAC-3′; primers used for detection of gB were 5′-GAAGATCCGCATGTCCTTCAG-3′ and 5′-AATCCGTCCAACATCTTGTCG-3′. Genome copy numbers were calculated using a standard curve generated with the pARK25 MCMV plasmid (a kind gift from Alec Redwood, University of Western Australia) with the limit of detection = 10 copies.

gB and IL-10 gene expression

IL-10 and gB were assayed by quantitative RT-PCR using a Mini Opticon and Platinum SYBR green mastermix reagent (Invitrogen). For IL-10 expression analysis, lung and spleen cells from mock or MCMV-infected mice were frozen on dry ice in Trizol reagent (Invitrogen). Thawed samples were homogenized and total cellular RNA was extracted and quantified. DNase-treated RNA was then used to synthesize cDNA with a TaqMan reverse transcription kit (Applied Biosystems). The primer sequences used for detection of IL-10 were: 5′-AGCATGGCCCAGAAATCAAG-3′ and 5′-CGCATCCTGAGGGTCTTCA-3′. For gB expression analysis, RNA was isolated from lung and spleen tissue (Qiagen); cDNA was then synthesised as described above and gB and actin were measured using the primers detailed above.

Virus reactivation assay

Virus-infected organs were divided into 3 parts and placed in separate wells in a 6-well plate containing 5ml (spleen) or 3ml (lung) D10 medium. Tissue was gently minced with the end of a 2ml syringe and, in the case of lung pieces, an additional 2ml D10 was then added. Tissue pieces were then cultured for 5 weeks; 4ml supernatant was collected weekly and replaced with 5ml fresh D10. Sonicated supernatant was then assayed for infectious virus by plaque assay as described previously (38).

Results

IL-10 limits memory CD8 T cell expansion during MCMV infection

In our C57BL/6 mouse model of MCMV infection, productive replication (data not shown) and late viral gene expression (Fig. S1A) was undetectable in the spleen and lung by 60 days post-infection. In contrast, viral DNA was present in both organs and virus could reactivate ex vivo in cultures of spleen and lung isolated as late as 110 days post-infection (Fig. S1B), thereby suggesting that MCMV persisted in these organs primarily in the form of latent virus. Despite the absence of detectable MCMV replication from day 60, expression of IL-10 in the spleen (but not lung) was increased compared with uninfected mice (Fig. 1A). Flow cytometric analyses revealed IL-10 protein production by splenic IFN-γ+IL-17− MCMV-specific CD4 T cells after peptide stimulation directly ex vivo (Fig. 1B). The frequency of IL-10-producing cells within MCMV-specific CD4 T cell populations was substantially higher than that observed in IFN-γ− CD4 T cells (3.7-8.3%, mean=6.3% versus 0.08-0.3%, mean=0.19%, p <0.01). Splenic B cells (CD19+) and monocytes/macrophages (CD11b+) derived from mice 90 days post-infection were also capable of producing IL-10 following ex vivo non-specific stimulation; in the case of CD11b+ cells, but not CD19+ cells, this MCMV-associated IL-10 production was significantly increased compared to cells derived from naïve mice (Fig 1C).

Figure 1. IL-10 limits memory CD8 T cell inflation during MCMV infection.

Figure 1

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(A) IL-10 expression in spleen extracts from naive C57BL/6 (wt) mice and wt mice infected for 90 days with MCMV was measured by quantitative RT-PCR and normalized to β-actin. Results represent the mean +/− SEM of 4 mice from 2 independent experiments. Significance is indicated by *, which represents p <0.05 with the Mann-Whitney U-test. (B) IFN-γ and IL-17 expression by CD4 T cells derived from spleens of wt mice 90 days post-infection (top panels) following ex vivo stimulation for 16 hours with (top left panel) or without (top right panel) m09, M25, m139 and m142 peptides. Values in quadrants represent %cytokine+CD4+ cells. Expression of IL-10 by IFN-γ+ peptide-specific CD4 T cells from wt (bottom left panel) and IL-10−/− (bottom right panel) mice. Quadrants were set using the fluorescence minus one (FMO) control for APC. Results are representative of 2 experiments, each comprising 4 mice per group. (C) IL-10 expression by splenic CD19+ and CD11b+ cells derived from naïve (closed bars) and MCMV-infected (day 60 post-infection; open bars) mice following ex vivo stimulation with LPS and PMA/ionomycin (CD19+) or LPS alone (CD11b+). Results are shown as the mean +/− SEM derived from 2 independent experiments, each comprising 4 mice per group. Ex vivo stimulation of B cells from MCMV-infected mice (day 90 post-infection) did not induce significant apoptosis (6.6 +/− 0.4% AnnV+) as compared with freshly isolated CD19+ cells (8.2 +/− 2.2% AnnV+). (D and E) Wt (closed circles) and IL-10−/− (open circles) mice were infected with MCMV and peptide-specific CD8 T cells were quantified by intracellular IFN-γ detection. (D) Numbers of CD8 T cells specific for M38, M45, m139 and IE3 on days 0, 7, 14, 30 and 90 post-infection in spleen (left) and lung (right). Results are expressed as mean +/− SEM cells/organ of 4-6 mice per group and each time-point represents 4-7 experiments. Significance is indicated by *, which represents p <0.05 with Student’s T-test. (E) Representative bivariate flow cytometry plot showing IFN-γ expression by IE3-reactive CD8 T cells in the spleens (top panels) and lungs (bottom panels) of wt (left panels) and IL-10−/− (right panels) mice at day 90 post-infection. The % CD8 T cells expressing IFN-γ is shown; gates were set using the FMO control for FITC. Results are representative of 7 experiments, each comprising 4-6 mice per group.

We hypothesised that IL-10 acts to inhibit systemic and mucosal antiviral T cell responses during MCMV infection. To test this, we measured the accumulation of both “stable” memory CD8 T cells specific for a peptide derived from the M45 protein that is immunodominant during acute infection and “inflationary” memory CD8 T cells specific for peptides derived from the M38, m139 and immediate-early 3 (IE3) proteins in both wild-type C57BL/6 and IL-10−/− mice. Expression of these viral proteins in infected cells is thought to occur during productive replication rather than viral latency (20, 44). During acute infection, there were comparable numbers of MCMV-specific CD8 T cells in the lungs and spleens of wild-type and IL-10−/− mice (Fig. 1D); furthermore, as infection progressed, the numbers of M45-specific memory CD8 T cells remained low in the absence of IL-10 (Fig. 1D). Consistent with previously published data (20), high frequencies of CD8 T cells specific for M38, m139 and IE3 were observed in wild-type mice at day 90 post-infection; strikingly, however, the corresponding frequencies at the same time-point were even higher in IL-10−/− mice (Fig. 1D). The accumulation of IE3-specific CD8 T cells was most notable, representing ~15% of all CD8 T cells in IL-10−/− mice at day 90 post-infection (Fig. 1E) and reaching up to 30% in some individual mice (data not shown).

IL-10 limits the oligoclonal expansion of IE3-specific CD8 T cells

To investigate further the profound expansion of IE3-specific CD8 T cells, we undertook a comprehensive and unbiased analysis of T cell receptor (TCR) gene usage within the cognate populations isolated directly ex vivo from both wild-type and IL-10−/− mice (Fig. 2 and, for complete data set, Fig. S2). In both sets of mice at day 90 post-infection, the IE3-specific TCR repertoires were oligoclonal and highly skewed towards the usage of only a few dominant clonotypes; this is consistent with previous studies of HCMV-specific CD8 T cell populations (42). Moreover, a diverse array of clonotypic structures was apparent in both wild-type and IL-10−/− mice, although with a strong bias towards TRBV16 usage (Fig. 2 and Fig. S2). Similar patterns were observed as late as day 282 post-infection, at which time IE3-specific CD8 T cells dominated the response in IL-10−/− mice (Figs. S3, S4 and S5). In addition, a substantial degree of differentially encoded inter-individual TCR sharing was apparent (Fig. 2 and Fig. S2, S4 and S5); this is consistent with recent studies demonstrating the role of convergent recombination in the generation of “public” clonotypes, which can affect the outcome of infection in certain circumstances (45, 46). Overall, these data suggest that the enhanced inflation of IE3-specific CD8 T cells in IL-10−/− mice is due to the expansion of clonotypes selected within the memory pool rather than the recruitment of greater numbers of cognate clonotypes from the naïve pool (47).

Figure 2. Oligoconal expansion of IE-3 specific CD8 T cells during MCMV infection.

Figure 2

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TCRB CDR3 amino acid sequences, TCRBV and TCRBJ usage, and the relative frequencies of individual clonotypes within viable CD3+CD8+IE3-tetramer+ cell populations isolated by flow cytometric sorting from wt (A) and IL-10−/− (B) mice at day 90 post-MCMV infection. Three representative mice are shown per group; colored boxes represent shared clonotypes that were detected in other individual mice. The full dataset is shown in Supplemental Figure S2; analyses from later time-points are shown in Supplemental Figure S4 (day 142 post-infection) and Supplemental Figure S5 (day 282 post-infection).

Enhanced MCMV-specific CD4 T cell and professional APC accumulation in IL-10−/− mice during MCMV infection

CD4 T cells promote MCMV-specific memory CD8 T cell expansion (48, 49). We therefore measured the accumulation of virus-specific memory CD4 T cell populations in wild-type and IL-10−/− mice, investigating previously described distinct CD4 T cell populations with specificities for 4 different viral proteins (m09, M25, m139, m142) that are associated with active viral replication (39). Interestingly, we found that enhanced CD8 T cell accumulation in IL-10−/− mice at day 90 post-infection was accompanied by a large increase in the number of IFN-γ-expressing CD4 T cells in the spleen (Fig. 3A and B) and lung (Fig. 3C and D) detected following ex vivo stimulation with MCMV-derived MHC class II-restricted peptides (Fig. 3A and C) or PMA and ionomycin (Fig. 3B and D). IL-10 can inhibit CD4 T cells by suppressing the function of professional APC (26, 27) and MCMV-induced IL-10 inhibits MHC class II expression (50). In accordance, we observed more splenic DCs (Fig. 3E and F), splenic inflammatory monocytes (Fig. 3G), and pulmonary macrophages (Fig. 3H) in IL-10−/− mice compared to wild-type mice. In contrast, B cell accumulation was comparable in spleens and lungs of wild-type and IL-10−/− mice (data not shown).

Figure 3. Elevated CD4 T cell accumulation at day 90 following MCMV infection of IL-10−/− mice.

Figure 3

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IFNγ-expressing CD4 T cells in the spleens (A and B) and lungs (C and D) of wt (closed bars) and IL-10−/− (open bars) mice at day 90 post-infection following stimulation with peptide (A and C) or PMA and ionomycin (B and D). Undetectable virus-specific CD4 T cells were expressed as an arbitrary value of 1 cell for statistical analyses. (E) Representative flow cytometric analyses of splenic CD11chiMHCII+ dendritic cells from wt (left) and IL-10−/− (right) mice at day 90 post-infection. Splenic CD11chiMHCII+ (F) and CD11b+CD11c−Gr1lo/−7/4+ monocytes (G), and lung CD11cintCD11b+Gr1lo/− F4/80+ macrophages (H) from wt (closed bars) and IL-10−/− (open bars) mice at day 90 post-infection were enumerated. Results represent mean +/− SEM of 2-3 independent experiments, each comprising 4 mice per group. Significance is indicated by *, which represents p <0.05 with Student’s T-test.

IL-10 inhibition of CD8 T cell memory occurs during acute and chronic/latent MCMV infection

MCMV-specific memory CD8 T cell populations are derived both from cells primed during acute infection and from more recent thymic emigrants (22). Given that IL-10 is expressed during acute MCMV infection (39, 51-53), and enhances CD4 (52) and, in some models, CD8 T cell responses (53) at this time, we next examined the time-point at which IL-10 exerts its inhibitory effect.

Blockade of IL-10R signalling during initial T cell priming using a monoclonal antibody at the time of infection did not significantly influence CD8 T cell numbers specific for M38 and m139 at day 96 post-infection (Fig. 4A). However, IE3-specific CD8 T cells, which are generally undetectable in the first week of infection (20, 22), were enhanced approximately two-fold at the day 96 time-point (Fig. 4A). This was accompanied by a transient expansion of MCMV-specific CD4 T cell numbers at day 7 (Fig. 4B) but not day 90 (data not shown) post-infection.

Figure 4. IL-10 inhibits memory CD8 T cells during acute and chronic/latent MCMV infection.

Figure 4

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(A and B) MCMV-infected wt mice were treated with IgG (closed bars) or αIL-10R (open bars) on day 0 and peptide-specific CD8 (A) and CD4 (B) T cells were enumerated at day 96 (A) and day 7 (B) post-infection. (C-E) Wt mice were treated with IgG or αIL-10R on days 60, 67, 74, and 81 post-infection. (C) Representative bivariate flow cytometry plots showing IFN-γ expression by CD8 T cells specific for M38, m139 and IE3 at day 90 post-infection from mice treated with IgG (top panels) or αIL-10R (bottom panels). Peptide-specific CD8 (D) and CD4 (E) T cells were enumerated at day 90 post-infection. MCMV-specific CD4 T cells were stimulated with pooled m09, M25, m139 and m142 peptides. Results are expressed as the mean +/− SEM of 2 independent experiments, each comprising 4 mice per group. Significance is indicated by *, which represents p <0.05 with Student’s T-test.

Next, we inhibited IL-10R signalling from day 60 post-infection. Importantly, CD8 T cell populations specific for M38, m139 and IE3 were all substantially increased (3-4 fold) following late IL-10R blockade (Fig. 4C and D), as were MCMV-specific CD4 T cells (Fig. 4E). In the case of IE3-specific CD8 T cells, this increased accumulation was more dramatic than that observed after early IL-10R blockade (Fig. 4A). Thus, IL-10 primarily limits memory T cell accumulation during the chronic/latent phase of infection.

CD27 expression is down-regulated by MCMV-specific CD8 T cells from IL-10−/− mice

In contrast to the phenotypic heterogeneity of the total CD8 T cell population (Fig. 5A), inflationary MCMV-specific memory CD8 T cells from wild-type mice were CD44hiCD62Llo (Fig. 5A), consistent with an effector memory status. Furthermore, these highly differentiated cells expressed high levels of KLRG-1 and low levels of CD27 (Fig. 5A). Memory CD8 T cells specific for MCMV from IL-10−/− mice were also CD44hiCD62Llo (Fig. 5A). Notably, however, the frequency of KLRG-1+CD27− MCMV-specific CD8 T cells was even higher in IL-10−/− mice (Fig. 5A and B) and down-regulation of CD27 was more pronounced (Fig. 5A and C). In addition, receptors for the homeostatic cytokines IL-7 and IL-15 were not up-regulated by CD8 T cells from IL-10−/− mice (data not shown). Collectively, these data suggest that IL-10 does not inhibit memory CD8 T cells via the regulation of inhibitory or cytokine receptor expression; furthermore, it seems that the enhanced expansion of inflationary MCMV-specific CD8 T cells in the absence of IL-10 is associated with more advanced differentiation.

Figure 5. IL-10−/− MCMV-specific CD8 T cells are highly differentiated.

Figure 5

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MCMV-specific CD8 T cells from spleens of wt and IL-10−/− mice were detected with cognate tetramers and co-expression of CD27, KLRG-1, CD44, and CD62L was examined. (A) Co-expression of CD27 and KLRG-1 (left panels), and CD62L and CD44 (right panels), by CD3+CD8+tetramer+ cells (colored dots) and total CD3+CD8+ cells (black/grey) from spleens of wt (left panels in each set) and IL-10−/− (right panels in each set) mice. (B and C) The frequencies of KLRG-1+CD27− CD3+CD8+tetramer+ cells (B) and the median fluorescence intensity (MFI) of CD27 expression by CD3+CD8+tetramer+ cells (C) was assessed. In IL-10−/− mice, the %KLRG-1+CD27− cells in the total CD3+CD8+ population was not significantly increased (wt = 23.5, IL-10−/− = 24.1; p = 0.91, nor was the CD27 MFI (wt = 2564.75, IL-10−/− = 1979.5; p = 0.223). Results are expressed as the mean +/− SEM of 3 experiments, each comprising 3-4 mice per group. Significance is indicated by *, which represents p <0.05 with Student’s T-test.

IL-10 restricts the accumulation of functional MCMV-specific CD8 T cells

We next hypothesized that IL-10 limits the accumulation of functional antiviral memory CD8 T cells. Hence, we examined cytokine production by MCMV-specific CD8 T cells. A low proportion (6-10%) of wild-type MCMV-specific IFN-γ+ CD8 T cells co-expressed IL-2 following ex vivo peptide stimulation and this was further reduced (2-4%) in IL-10−/− mice (Fig. 6A). In contrast, IL-10 deficiency increased both the proportion (Fig. 6A) and the total number (Fig. 6B) of MCMV-specific IFN-γ+ CD8 T cells capable of co-expressing the antiviral cytokine TNF. We also quantified recent degranulation based on cell surface CD107a mobilization and observed greater numbers of CD107a+ MCMV-specific CD8 T cells in the spleens and lungs of IL-10−/− mice compared to wild-type mice after ex vivo peptide stimulation (Fig. 6C). Importantly, IL-10R blockade in wild-type mice from day 60 post-infection also led to an increase in the numbers of virus-specific CD8 T cells that expressed TNF (Fig. 6D) and degranulated (Fig. 6E) in response to peptide stimulation. Collectively, these data suggest that IL-10 restricts the accumulation of functional memory CD8 T cells.

Figure 6. IL-10 limits the accumulation of functional antiviral CD8 T cells during MCMV infection.

Figure 6

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TNF, IL-2 and CD107a expression by IFN-γ+ MCMV peptide-specific CD8 T cells from wt and IL-10−/− mice (A-C) and wt mice treated with IgG or αIL-10R (D and E) was assessed at day 90 post-infection. (A) Representative bivariate flow cytometry plots showing TNF versus IL2 expression by IFN-γ− CD8 T cells (control) and IFN-γ+ peptide-specific splenic CD8 T cells from wt (top panels) and IL-10−/− (bottom panels) mice. Results are representative of 4 experiments, each comprising 4-5 mice per group. (B-E) Numbers of peptide-specific CD8 T cells expressing TNF (B and D) and surface CD107a (C and E) in the spleens (left panels) and lungs (right panels) of wt (closed bars) and IL-10−/− (open bars) mice (B and C) and wt mice treated with IgG (closed bars) or αIL-10R (open bars) on days 60, 67, 74 and 81 post-infection (D and E). All results are representative of 3 experiments and show the mean+/− SEM of evaluations conducted with 4-5 mice per group. Significance is indicated by *, which represents p <0.05 with Student’s T-test.

Latent virus load is reduced in the absence of IL-10R signalling

To determine whether the increased CD8 T cell responses in IL-10−/− mice reduced latent viral load, MCMV genome content in the lung and spleen was examined. No significant differences were observed at the peak of the primary T cell response (day 7 post-infection, data not shown) or at day 60 post-infection (Fig. 7A). Critically, however, viral DNA load was significantly reduced in both the spleens and lungs of IL-10−/− mice at day 90 post-infection (Fig. 7B). Importantly, late (from day 60 onwards) blockade of IL-10R in wild-type mice also led to a reduction in viral DNA load in spleen and lung at this time (Fig. 7C).

Figure 7. IL-10 increases latent MCMV load during infection.

Figure 7

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Genomic DNA was isolated from the spleens and lungs of wt (closed bars) and IL-10−/− (open bars) mice at days 60 (A) and 90 (B) post-infection. (C) Infected wt mice were treated on days 60, 67, 74 and 81 with IgG (closed bars) or αIL-10R (open bars) and genomic DNA was isolated from the spleens and lungs at day 90 post-infection. MCMV gB was detected by quantitative PCR and data expressed as genome copy number per 100ng genomic DNA; horizontal dashed lines depict the lower limit of detection. Results are representative of 2 independent experiments and show the mean +/− SEM of evaluations conducted with 5-15 mice per group. Significance is indicated by *, which represents p <0.05 with the Mann-Whitney U-test.

Discussion

Herein, we report that IL-10 suppresses CMV-specific memory T cell inflation. The key findings of this study were: (i) chronic/latent MCMV infection was associated with IL-10 production by macrophages and virus-specific IFN-γ-expressing CD4 T cells; (ii) mice deficient in IL-10 exhibited a profound increase in MCMV-specific CD8 T cell accumulation and antiviral cytokine production; (iii) IL-10 suppressed the oligoclonal expansion of memory CD8 T cells; (iv) MCMV viral genome load was reduced in IL-10−/− mice; and, (v) the enhanced memory CD8 T cell expansion and reduced viral load observed in IL-10−/− mice was recapitulated by delayed IL-10R blockade in wild-type mice. Collectively, these data demonstrate that IL-10 restricts the accumulation of functional MCMV-specific memory T cells during MCMV infection.

In vivo, MCMV infection leads to chronic activation of APC (54) and the generation of highly differentiated, activated memory CD8 T cells (22, 24); these observations suggest that, after the resolution of acute infection, mice harbour non-replicating, reactivating and, possibly, low levels of chronically replicating CMV that drives the immune activation (55-57). In the present study, 3.7-8.3% of splenic IFN-γ+ MCMV-specific memory CD4 T cells from MCMV-infected mice expressed IL-10. The production of IL-10 by Th1-like cells is triggered by chronic stimulation in parasitic infections (58, 59), and prolonged stimulation of HCMV-specific CD4 T cells in vitro induces IL-10/IFN-γ co-expression (60). Similarly, we observed that MCMV infection led to an increased frequency of IL-10 expressing CD11b+ cells. Although we cannot exclude the possibility that MCMV may encode a protein(s) that selectively induces IL-10 expression by these cell subsets, our data imply that IL-10 expression by MCMV-specific CD4 T cells and monocytes/macrophages is a consequence of chronic activation which, in this model, is likely to be provided by persistent viral reactivation from latency and, possibly, undetectable chronic replication.

B cells are a significant source of IL-10 during acute systemic MCMV infection (53). Although chronic/latent infection did not increase the proportion of IL-10-producing CD19+ cells in the spleen as compared to naïve mice, we cannot exclude the possible importance of this cell subset. Intracellular cytokine staining is a relatively insensitive technique and may therefore underestimate the frequency of IL-10-producing CD19+ cells. Furthermore, given that ~50% of splenocytes were CD19+ at day 90 post-infection (unpublished observation), even the small percentage of CD19+ IL-10 producers suggested by our data could have a significant impact on antiviral immunity.

Interestingly, although IL-10 expression was detected in the lungs of mice at day 90 post-infection, this was not increased compared to naïve controls (data not shown). Whether this reflects the small numbers of virus-specific CD4 T cells in the lungs of chronic/latently infected mice in our model, or the presence of IL-10-producing interstitial macrophages (61) in both uninfected and infected mice, is not clear.

The amplification of CD8 T cell inflation in IL-10−/− mice was striking and maintained 10 months post-infection without causing overt signs of disease (weight loss and cachexia) or pathology (unpublished observation). In some models, IL-10 directly inhibits CD8 T cells (62, 63), whereas in other situations IL-10 actually promotes primary (64) and memory CD8 T cell responses (64, 65). In our system, inflationary CD8 T cells expressed low levels of IL-10R (data not shown), thereby suggesting that these cells might be refractory to direct IL-10-mediated effects. Interestingly, enhanced CD8 T cell responses in IL-10−/− mice were accompanied by increased numbers of CD4 T cells and APC. Inflationary CD8 T cell responses are partially dependent upon CD4 T cells (48, 49), thereby implying that elevated CD4 T cell help in IL-10−/− mice promoted CD8 T cell expansion. Interestingly, IE3-specific CD8 T cells, which are more sensitive to CD4 depletion than other memory CD8 T cells (49), were preferentially expanded in IL-10−/− mice. Collectively, these data suggest that IL-10 limits memory CD8 T cell expansion via the abrogation of effective CD4 T cell help.

Expansion of CD8 T cell numbers was not observed 7 days post-infection, as reported previously (52). Primary MCMV-specific CD8 T cell responses occur independently of CD4 T cell help (48, 49), suggesting that IL-10-mediated inhibition of CD4 T cells only limits memory CD8 cells. Interestingly, however, B cell-derived IL-10 inhibits primary CD8 T cell responses following sub-cutaneous infection (53). Whether these differences reflect the different mouse strains or infection routes used in this study, variation in how peptide-specific CD8 T cells were enumerated (percentages versus total numbers of splenic CD8 T cells) or the fact that we housed our IL-10−/− mice in specific pathogen-free conditions (and subsequently did not observe colitis in aged naïve mice or mice infected with MCMV for 14 months; data not shown), is unclear.

The increased expansion of CD8 T cells in IL-10−/− mice was not due to altered expression of cytokine receptors (CD122, CD127) or the absence of inhibitory molecules (KLRG-1). However, it is known that virus-specific CD8 T cells down-regulate CD27 during latent MCMV infection (24, 66), and we found that the expression of this surface marker was further reduced in IL-10−/− mice. One possible functional consequence of CD27 down-regulation in this study was reduced IL-2 production in response to antigen stimulation; this is consistent with previous studies linking cellular differentiation to the loss of IL-2 expression (67, 68) and suggests a role for CD4 T cell help in the maintenance of polyfunctional memory CD8 T cells (69). Furthermore, highly differentiated CD8 T cells express high levels of granzyme and perforin (66, 70, 71) thereby suggesting that these cells are cytotoxic. The increased accumulation in IL-10−/− mice of CD8 T cells that degranulate more readily on cognate antigen encounter further supports the conclusion that IL-10 restricts MCMV-specific cytotoxic T cell responses.

In chronic LCMV infection, dysregulated TNF production by CD8 T cells is rescued in IL-10−/− mice (33). However, in MCMV infection, virus-specific CD8 T cells sustain pro-inflammatory cytokine expression during latency (22). Indeed, 60-70% of virus-specific IFN-γ-producing CD8 T cells in wild-type mice co-expressed TNF. Therefore, enhanced TNF production by IL-10−/− CD8 T cells probably reflects increased activation rather than reversal of defective function. Furthermore, CD8 T cell functionality was not enhanced on a per cell basis following IL-10R blockade in wild-type mice (data not shown), highlighting that the qualitative restriction of CD8 T cell function by IL-10 was comparatively small as compared to the large effect on T cell numbers. Collectively, these data suggest that IL-10 predominately regulates inflationary CD8 T cell responses by inhibiting cellular accumulation at sites of viral infection.

Importantly, MCMV burden was reduced in IL-10−/− mice and wild-type mice treated with an IL-10R blocking antibody. Expanded CD8 T cells in both situations were reactive to several viral proteins expressed during active replication, thereby implying that these cells do not directly recognize latently infected cells. Of particular interest was the expansion of IE3-specific CD8 T cells. Immediate early (IE) genes are expressed during the early stages of viral replication, and IE3 is a check-point gene involved in the maintenance of latency (72). Patrolling IE-specific CD8 T cells may detect early reactivation events (73). It is therefore possible that the increased frequency of antigen recognition by IE3-specific cells may contribute to the more dramatic effect of IL-10 on this population. In support of this hypothesis, latent MCMV infection of BALB/c mice induces the inflation of IE1-specific CD8 T cells (19, 21) that terminate viral reactivation in vivo (74). Alternatively however, it is conceivable that enhanced memory T cell responses in the absence of IL-10R signalling may inhibit low levels of undetectable chronic replication which, in turn, restricts the establishment of latent infection in the spleen and lung.

The exact mechanism by which expanded CD8 T cells reduced viral load following IL-10 gene deletion or IL-10R blockade is also unclear. Both approaches led to enhanced numbers of CD8 T cells in the spleen and lung that could degranulate efficiently; in conjunction with the reduced levels of CD27 expression, this implies an enhanced capability to kill cells harbouring reactivating and/or replicating virus. Indeed, elevated TNF secretion by CD8 T cells in the absence of IL-10R signalling may promote splicing of IE3 transcripts and subsequent viral reactivation (75). In this situation, which could be implied by the preferential expansion of IE3-specific CD8 T cells in IL-10−/− mice, increased numbers of cytotoxic T cells would detect and kill cells harbouring this reactivating virus. Alternatively, increased accumulation of T cells expressing IFN-γ and TNF might directly inhibit late gene expression during viral reactivation and/or replication (76-78), and may also activate secondary antiviral mechanisms mediated by other cell types, such as macrophages and NK cells (79-81).

The factors that determine whether a MCMV-specific CD8 T cell population inflates are currently unknown. However, the observation that inflationary but not stable memory CD8 T cells are enhanced in IL-10−/− mice implies that IL-10 is not a determining factor in this process. Rather, IL-10 appears to limit the expansion of memory CD8 T cells that are already dividing during chronic/latent infection.

The data presented herein provide evidence that, as suggested previously (37, 82), IL-10 orthologs expressed by herpesviruses including HCMV (34) and Epstein Barr virus (83) may function to preserve latent viral load within the host. It should be noted, however, that MCMV does not encode an obvious IL-10 ortholog, and the kinetics and locality of expression of host and viral IL-10 proteins may differ in vivo. Furthermore, HCMV (84) and Epstein Barr virus (85) IL-10 orthologs promote B cell growth and differentiation, whereas in our model we observed no effect of murine IL-10 on B cell accumulation. Although we hypothesize that the increased numbers of CD4 T cells in IL-10−/− mice may compensate for the absence of direct IL-10R signalling in B cells, we cannot exclude the possibility that mammalian and viral IL-10 proteins could function differently in vivo. Irrespective, our data clearly highlight the previously unappreciated role for mammalian IL-10 in the regulation of memory T cell inflation in vivo and suggest that this immune regulatory pathway could be manipulated to promote the accumulation of functional antiviral memory T cells.

Supplementary Material

Sup Fig Legend
Sup Figs

Acknowledgements

The authors wish to thank Awen Gallimore, Andrew Godkin, Gavin Wilkinson, Phil Taylor and Ann Ager for helpful discussion, Rhonda Cardin for advice regarding the in vitro viral reactivation assay, and Andrea Loewendorf and Alec Redwood for advice on MCMV genome detection.

Footnotes

2

This work was supported by the Wellcome Trust and the Medical Research Council (MRC); I.R.H. is a Wellcome Trust Research Career Development Fellow, D.A.P. is an MRC Senior Clinical Fellow, M.F.Q. is a Marie Curie International Outgoing Fellow and M.A.S. is a Cardiff University I3-Interdisciplinary Research Group/MRC student.

3

Abbreviations: HCMV, human CMV; MCMV, mouse CMV; vIL-10, viral IL-10

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