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The Journal of Immunology, 2007, 179, 7325 -7333
Copyright © 2007 by The American Association of Immunologists, Inc.
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Murine Gammaherpesvirus-68 Infection Alters Self-Antigen Presentation and Type 1 Diabetes Onset in NOD Mice1

Katherine A. Smith, Stacey Efstathiou and Anne Cooke2

Department of Pathology, University of Cambridge, Cambridge, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Recent research in line with the "hygiene hypothesis" has implicated virus infection in the delay or prevention of autoimmunity in murine models of type 1 diabetes such as the NOD mouse. We found that intraperitoneal or intranasal infection of NOD mice with the murine gammaherpesvirus-68 (MHV-68) significantly delayed diabetes onset in an age-dependent manner. The acute phase following intraperitoneal infection was associated with significantly reduced trafficking of autoreactive BDC2.5NOD CD4+ T cells to the pancreas but not the pancreatic lymph node (PLN); this was not as a result of MHV-68 M3 pan-chemokine binding protein expression. Autoreactive BDC2.5NOD CD4+ T cells within the PLN of MHV-68 infected mice were significantly more naive and proliferated to a lesser extent than those cells within the PLN of uninfected mice. These changes in autoreactive CD4+ T cell activation were associated with reduced dendritic cell endocytosis and soluble Ag presentation but were not as a result of virally induced IL-10 or changes in Ag-specific regulatory T cell populations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Virus infection has historically been associated with the onset or exacerbation of autoimmune diseases such as type 1 diabetes (T1D).3 However, studies of viral infection in murine models of T1D have demonstrated that certain viruses can delay or prevent disease onset (1, 2, 3, 4, 5, 6). Many of these reports do not elucidate the mechanisms by which these diverse viruses suppress T1D due to the complexity of autoimmune disease onset and the host response to virus infection.

Murine gammaherpesvirus-68 (MHV-68) is a well characterized murine gammaherpesvirus, which has many immunomodulatory activities that might enable it to alter T1D onset. Following i.p. infection, MHV-68 undergoes acute infection, followed by the establishment of life-long latency. During both phases, MHV-68 expresses a number of factors known to influence the host immune response to infection. These include MK3, which reduces anti-viral CD8+ T cell recognition (7) and M3, which binds and blocks the function of CXC and CC chemokines (8, 9). The chemokine binding decoy receptor, M3, has been shown to reduce the inflammatory pathology in 12-O-tetradecanoylphorbol-13-acetate-treated D6-deficient mouse skin (10) and also to inhibit chemokine-induced recruitment of monocytes, dendritic cells (DCs), and lymphocytes into the pancreas of transgenic mice (11). In addition to the virally encoded immune modulators expressed by MHV-68, this virus also inhibits the host immune response by inducing IL-10 production (12). This ability of MHV-68 to modify the host immune response, coupled with the availability of mutant virus lacking immunomodulatory functions or with altered patterns of infectivity enabled a number of focused questions to be asked about how this virus might modify diabetes onset in NOD mice. As a result of data showing that particulate, as well as cell-associated Ags, from the peritoneal cavity are presented to CD4+ and CD8+ T cells within the pancreatic lymph nodes (PLNs) of adult mice (13), we have used the i.p. route of MHV-68 infection to deliver the virus directly to the PLN.

We find that i.p. or intranasal (i.n.) MHV-68 infection can significantly delay and decrease diabetes incidence in 8- and 9-wk-old female NOD mice. Maintenance of virus latency, the expression of the immune-evasion gene M3, viral induction of host IL-10, and preferential regulatory CD4+ T cell induction were not associated with T1D delay. The acute phase of MHV-68 following i.p. infection is associated with a reduction in DC sampling, processing and loading of self-Ag onto MHC class II and the subsequent activation of autoreactive CD4+ T cells in the PLN of NOD mice. When occurring at a critical threshold of autoreactive CD4+ T cell accumulation within the PLN, the presence of MHV-68 infection can significantly reduce the trafficking of autoreactive CD4+ T cells to the pancreas, decreasing the incidence of diabetes in NOD mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Virus and mice

MHV-68 was propagated and titered using baby hamster kidney-21 (BHK-21) fibroblasts. Recombinant viruses containing disruptions of the M3 and ORF73 gene have been described previously (14, 15). Concentrated virus stocks were free from endotoxin <10EU/ml and were diluted at least 1000-fold in endotoxin free PBS before administration to animals.

NOD, BDC2.5NOD, and GFP.BDC2.5NOD mice were bred and maintained under specific pathogen-free barrier conditions. BALB/c mice were obtained from Harlan. All animal work was conducted under home office project license regulations after approval by the Ethical Review Committee of the University of Cambridge, U.K.

Female NOD mice were infected with 104 plaque-forming units (PFU) MHV-68 i.p. or i.n. as indicated at differing ages and monitored for the presence of urinary glucose using Diastix (Bayer). Mice were considered diabetic if they had a urinary glucose concentration of 12 mM or above on two occasions.

Infectious virus was assayed on BHK-21 cells following homogenization and freeze-thaw disruption of tissues. Latent virus from the spleen was assessed by infectious center assay (16). For this assay, single-cell suspensions of splenocytes were cocultured with BHK-21 cells for 4–5 days. Monolayers were then fixed with 10% formal saline and stained with toluidine blue. Plaques were counted using a plate microscope.

Investigation of autoreactive T cell fate

Single-cell suspensions of splenic lymphocytes from BDC2.5NOD or GFP.BDC2.5NOD mice were prepared and RBC lysed in ammonium chloride buffer. For CFSE labeling, cells were incubated in PBS with 5 µm CFSE (Molecular Probes). For transfers, 5 x 107 cells were injected into a lateral tail vein of mice. Mice were sacrificed 72 h later; spleens, pancreata, pancreatic, and mesenteric lymph nodes were harvested and single-cell suspensions were prepared as described previously (17). In brief, pancreata were torn into pieces in cold PBS containing 5% FCS, 56 mM glucose (Sigma-Aldrich), and Complete Mini Protease inhibitors (Roche). The tissues were washed twice in cold PBS, before digestion in 2 ml prewarmed PBS containing 15% FCS and Liberase CI (Roche) in a shaking incubator. After digestion, tissues were washed in complete medium and cell suspensions prepared by forcing through a 70-µm cell strainer. Suspensions were left to settle to remove stromal debris, supernatants were removed and washed in complete medium before Ab staining and analysis.

Flow cytometric analysis

Nonspecific Ab binding was blocked by incubation with mAb clone 2.4G2 (ATCC number HB-197). T cells were stained by standard methods using rat anti-mouse reagents CD4-PerCP, CD44-PE, CD62L-APC, CD69-PE, CD25-APC (all BD Pharmingen), and CCR7-PE (AbCam). DCs were stained with a range of Abs including CD11c-APC, CD80-PE, CD86-PE, CD83-PE, CD8-PerCP, CD4-PerCP, and H2Db-PE, OX-6-biotin with Streptavidin-PE for NOD DCs. Foxp3 staining was conducted according to protocols provided by eBiosciences. Intracellular cytokine staining with anti-IFN-{gamma}-PE (XMG1.2), anti-IL-10-PE (JES5–16E3), or appropriate isotype control (BD Pharmingen) was performed as previously (17). All analysis was conducted using a BD LSR 1 flow cytometer (BD Biosciences).

Analysis of virally induced IL-10 on autoreactive T cell fate

Anti-IL-10R (1B1.3a) and a rat IgG1 control Ab (MAC221) were obtained from Dr. K. Moore (DNAX, Palo Alto, CA) and Dr. G. Butcher (Babraham Institute, Cambridge, MA) respectively. After i.p. infection, 8-wk-old female NOD mice were treated with 200 µg 1B1.3a, MAC221, or PBS control i.p. on day 3 and 5 postinfection and directly preceding BDC2.5NOD splenocyte transfer (day 7 postinfection.). This dose of 1B1.3a was shown to be effective in vivo (18); endotoxin levels were <1 EU/mg protein. Autoreactive T cell populations were transferred and tracked as described above.

Phagocytosis of fluorescent microspheres and dendritic cell purification

Eight-wk-old female NOD mice were injected i.v. with 3.64 x 1010 Fluoresbrite YG carboxylate microspheres (0.5 µm; Polysciences) 8 days postinfection. Salmonella typhimurium LPS was used as a positive control for systemic activation of dendritic cells (19) and 5 µg was injected i.v. 16 h before microsphere transfer. Three hours after microsphere transfer, spleens were collected and DCs were isolated by positive selection or analyzed by flow cytometry.

Splenic DCs were prepared using CD11c microbeads (Miltenyi Biotec). In brief, spleens were cut into fragments and digested in medium containing Liberase CI (Roche). DC-T cell complexes were then disrupted by the addition of EDTA (0.1 M (pH 7.3)). Splenic fragments were forced through a nylon cell strainer (BD Biosciences) and the resulting suspension was washed. Cell pellets were resuspended in cold buffer (PBS, 0.5% BSA, 2 mM EDTA) with CD11c microbeads, before being washed in buffer. Cells were then passed through LS columns (Miltenyi Biotec) for the selection of high purity CD11c+ cells as determined by FACS.

Ex vivo Ag presentation assays

Positively selected splenic DCs were seeded at 2 x 104, 2 x 105, and 2 x 106 cells/well. OVA protein (Sigma-Aldrich) or BDC2.5 peptide; sequence Ac-RTRPLWVRME-NH2, (20) was added at 40 mg or 1 µg/ml respectively and titrated 10-fold in triplicate before incubating for at least an hour at 37°C/5%CO2. After washing with medium 2 x 105 CFSE-labeled DO11.10 or BDC2.5NOD RBC lysed splenocytes were added per well and incubated for 24 h to analyze IL-2 in the supernatant and 72 h to analyze DO11.10 or BDC2.5NOD CD4+ T cell proliferation.

Statistics

Diabetes incidence was compared using the Logrank Test. Data gained from FACS analysis was compared using a nonparametric Mann-Whitney U test (two-tailed 95% confidence levels). A p value < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
MHV-68 infection can significantly decrease and delay T1D onset in 8-wk-old female NOD mice

We first determined whether i.p. MHV-68 infection could alter T1D onset in the NOD mouse by infecting female mice of differing ages with 104 PFU virus. Virus infection reproducibly and significantly delayed and decreased diabetes onset in female mice aged 8 or 9 wk old but not 7 wk old (Fig. 1A). A similar time-dependent delay in diabetes onset was also seen when mice were infected i.n. (Fig. 1B). The reproducibility of this time dependent delay and decrease in diabetes onset was confirmed at the 8 and 9 wk time-points (Fig. 1C) following i.n. and i.p. infection respectively. To establish a mechanism for this diabetes delay we examined whether homing of autoreactive CD4+ T cells from the BDC2.5NOD mouse to the pancreas and PLN of NOD mice was affected during lytic and latent phases of virus infection.


Figure 1
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  		FIGURE 1. Intraperitoneal (A) and (B) i.n. infection with MHV-68 significantly reduces and delays T1D onset in NOD mice in an age-dependent manner. A, 7 (i), 8 (ii), and 9 (iii) or (B) 4 (i), 6 (ii), 8 (iii), and 10 (iv) wk old female NOD mice were injected with 104 PFU MHV-68 ({blacktriangleup}) or PBS ({blacksquare}) and monitored for glucosuria (n = 10 mice/group). C, Data from repeat experiments each with internal PBS controls demonstrating a significant delay and decrease in diabetes following i.p. infection with MHV-68 at 9 wk old (i) and following i.n. infection with MHV-68 at 8 wk old (ii) (n = 12 mice/group).

 
Autoreactive CD4+ T cell homing to the pancreas is significantly reduced in NOD mice during acute phase MHV-68 infection and not during maintenance of viral latency

Following a self-limiting acute phase of infection, MHV-68 is known to establish life-long latency in the infected host. We were unable to detect infectious virus within the PLN and pancreas during the acute phase of infection (data not shown). However, latent virus could readily be detected in the spleen and PLN by infectious centre assay (Fig. 2A). Latent virus could not be detected in the pancreas of infected NOD mice. The inability to detect either infectious or latent virus in the pancreas indicates that this virus is not particularly pancreatrophic (data not shown).


Figure 2
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  		FIGURE 2. Trafficking of autoreactive CD4+ T cells to the pancreas is significantly reduced during acute MHV-68 infection 7–10 days postinfection (B) but not during latency (C). A, Spleen or PLN cells from 8-wk-old female NOD mice infected i.p. with 104 PFU MHV-68 were incubated with 3 x 105/well BHK-21s for 4 days at 37°C/5% CO2 over a range of time-points. BHK-21 monolayers were fixed, plaques counted, and expressed as Log10 PFU/107 splenocytes or per organ. B and C, Two x 107 CFSE-labeled BDC2.5NOD splenocytes were transferred to NOD mice day 7 (B) or day 28 (C) after i.p. injection with 104 PFU MHV-68 ({blacktriangleup}) or 200 µl PBS as a control ({blacksquare}). Pancreata were harvested 72 h posttransfer, labeled and the percentages of transferred autoreactive CD4+CFSE+ population were analyzed by FACS. The ratio of transferred CD4+CFSE+ cells to the endogenous CD4+ population was also determined by FACS. ns = not significant.

 
We transferred CFSE-labeled BDC2.5NOD splenocytes into mice on days 7 and 28 postinfection to analyze the impact on trafficking of these cells to the pancreas during periods of primary and latent infection. Trafficking was significantly reduced in MHV-68 infected mice 7 days postinfection (Fig. 2B) but was not affected in mice by day 28 after infection, a time associated with viral latency (Fig. 2C). MHV-68 ORF73 functions in the episomal maintenance of latent virus genomes and plays a crucial role in the maintenance of latency in the host without affecting lytic viral replication (15). To confirm that the reduction in trafficking of BDC2.5NOD CD4+ T cells was a consequence of acute but not latent MHV-68 infection, we transferred GFP.BDC2.5NOD splenocytes to mice 7 days after infection with wild-type MHV-68, a latency deficient recombinant virus containing a disruption of ORF73 (FS73) and uninfected mice. Using this approach, we were able to demonstrate that the reduced trafficking of autoreactive CD4+ T cells was unaffected by a failure to maintain viral latency (Fig. 3). When 8-wk-old female NOD mice were infected with the mutant and revertant (FS73R) virus we found that the incidence of spontaneous T1D was significantly reduced (p = 0.0151) with both virus strains compared with the uninfected control (58% FS73, 58% FS73R, and 100% PBS control by 32 wk old). These observations demonstrate that the acute phase of MHV-68 infection, but not the establishment and maintenance of a normal latent load can reduce autoreactive CD4+ T cell trafficking to the pancreas and result in a reduction in T1D.


Figure 3
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  		FIGURE 3. Significant reduction in the trafficking of autoreactive CD4+ cells to the pancreas is not influenced by maintenance of virus latency or by expression of MHV-68 M3. Two x 107 GFP.BDC2.5NOD splenocytes were transferred to 8-wk-old female NOD mice day 7 after i.p. injection of 104 PFU wild-type MHV-68 ({blacktriangleup}), a recombinant virus containing a disruption of the M3 gene (M3KO ({blacktriangledown})), the revertant of the recombinant virus (M3R ({diamondsuit})), a recombinant virus containing a frameshift mutation of ORF73 (FS73(•)) or 200 µl PBS as a control ({blacksquare}). Pancreata were harvested 72 h posttransfer and labeled and the percentages of transferred autoreactive CD4+CFSE+ population were analyzed by FACS. The ratio of transferred CD4+CFSE+ cells to the endogenous CD4+ population was also determined by FACS. Data is representative of three experiments performed with ≥3 mice/group.

 
Chemokine gradients are known to mediate the trafficking of a number of immune cell populations and because MHV-68 is known to express a secreted pan-chemokine binding protein M3 during its lytic and latent phases of replication it was of interest to determine whether the M3 product was influencing autoreactive CD4+ T cell trafficking. We transferred GFP.BDC2.5NOD splenocytes to NOD mice infected with wild-type MHV-68, a recombinant virus containing a disruption of the M3 gene (M3KO) and the corresponding revertant (M3R). The significantly reduced trafficking of the autoreactive GFP.BDC2.5NOD CD4+ population was unchanged in the absence of the M3 gene (Fig. 3). These observations indicate that the pan-chemokine binding protein M3 does not influence autoreactive CD4+ T cell trafficking during acute phase MHV-68 infection or diabetes onset.

Acute MHV-68 infection is associated with lymphadenopathy, changes in cellularity, and reduced proliferation and activation of autoreactive CD4+ T cells within the PLN

Autoreactive T cell activation is thought to occur within the draining lymph node in organ-specific autoimmune disease. To investigate the events preceding autoreactive CD4+ T cell trafficking to the pancreas, we analyzed the trafficking of transferred CFSE-labeled BDC2.5NOD splenocytes to the PLN as well as the activation and proliferation of these cells in an environment associated with MHV-68 infection. Trafficking and retention of autoreactive CD4+ T cells to the PLN was not altered during primary MHV-68 infection or at times associated with latency establishment in the PLN (Fig. 4B), although infection resulted in an increase in lymph node cellularity (Fig. 4A), total CD4+ number (Fig. 4C), and total CD11c+ number (Fig. 4D). The proliferation (Fig. 5A) and activation (Fig. 5B) of autoreactive CD4+ T cells within the PLN was significantly reduced during acute infection. Changes in the percentage of CD4+ T cell activation markers were seen for CD62L, CD44, the lymph node retention marker, and chemokine receptor CCR7 but not for the chemokine receptor CXCR3 (Fig. 5B). These data suggest that primary infection with MHV-68 and/or initial seeding of latently infected cells in the PLN can alter autoreactive CD4+ T cell activation and the initiation of diabetes in the NOD mouse.


Figure 4
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  		FIGURE 4. Acute MHV-68 infection does not alter autoreactive CD4+ T cell trafficking to the PLN but does alter PLN total cell number including endogenous CD4+ T cell and CD11c+ DC number. Two x 107 CFSE-labeled BDC2.5NOD splenocytes were transferred to 8-wk-old female NOD mice day 7 after i.p. injection with 104 PFU MHV-68 ({blacktriangleup}) or 200 µl PBS as a control ({blacksquare}). Pancreatic lymph nodes were harvested and counted 72 h posttransfer. Total cell numbers (A), transferred autoreactive CD4+CFSE+ cell numbers (B), endogenous CD4+ T cell numbers (C), and endogenous CD11c+ dendritic cell numbers (D) are shown. ns = not significant.

 

Figure 5
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  		FIGURE 5. The proliferation (A) and activation status (B) of autoreactive CD4+ T cells within the PLN is significantly altered 7–10 days postinfection. Two x 107 CFSE-labeled BDC2.5NOD splenocytes were transferred to 8-wk-old female NOD mice day 7 after intraperitoneal injection of 104 PFU MHV-68 ({blacktriangleup}) or 200 µl PBS as a control ({blacksquare}). PLN were harvested 72 h posttransfer, labeled, and proliferation of transferred autoreactive CD4+CFSE+ in the PLN was analyzed by CFSE dilution. The activation status of transferred autoreactive CD4+CFSE+ cells was assessed by expression of CD62L, CD44, the lymph node retention marker CCR7, and the chemokine receptor CXCR3. Data represent several experiments with ≥4 mice/group. ns = not significant.

 
Changes in autoreactive T cell activation are not associated with an increase in Ag-specific regulatory T cells or the induction of virally induced IL-10

A number of factors are thought to alter autoreactive T cell activation and subsequent autoimmune disease development. Ag-specific regulatory T cells have long been implicated in dampening down inflammatory responses by reducing CD4+ T cell activation; recent publications have demonstrated that MHV-68 can limit CD4+CD25+ activity and decrease Foxp3 expression in splenic CD4+ T cells (21). We therefore analyzed changes in the Foxp3 expression of transferred CFSE-labeled BDC2.5NOD CD4+ T cells and of endogenous CD4+ T cells and quantified the numbers of CD4+ regulatory T cells within the PLN of MHV-68 infected and uninfected NOD mice. There was a reproducibly significant reduction in the percentages of BDC2.5NOD CD4+ T cells expressing Foxp3 within the PLN of MHV-68 infected NOD mice (Fig. 6A), however, the percentage of endogenous PLN CD4+ T cells expressing Foxp3 was not affected by i.p. MHV-68 infection (Fig. 6B).


Figure 6
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  		FIGURE 6. There is no preferential expansion of Foxp3+ expressing autoreactive CFSE+ and endogenous PLN CD4+ T cells (A–D), or any changes in the percentage of Foxp3-expressing pancreatic CD4+ T cells during acute MHV-68 infection (E). Two x 107 CFSE-labeled BDC2.5NOD splenocytes were transferred to 8-wk-old female NOD mice day 7 after i.p. injection of 104 PFU MHV-68 ({blacktriangleup}) or 200 µl PBS ({blacksquare}). PLN and pancreata were harvested 72 h posttransfer, counted, and stained for CD4 and Foxp3. The percentage of Foxp3-expressing transferred CFSE+ (A) and endogenous CFSE– (B) PLN CD4+ cells was determined by FACS. Numbers of autoreactive CD4+CFSE+Foxp3+ (C) and the proportion of endogenous CD4+CFSE–Foxp3+ to CD4+CFSE–Foxp3+ (D) cells were determined from total cell counts. The percentage of Foxp3 expressing endogenous CD4+ cells in the pancreas was also determined by FACS (E). ns = not significant.

 
The total number of the transferred BDC2.5NOD CD4+ Foxp3+ T cells within the PLN was not altered during acute MHV-68 infection (Fig. 6C). The numbers of both CD4+Foxp3+ and CD4+Foxp3– cells increased in the endogenous population and the ratio of endogenous CD4+Foxp3+ to CD4+Foxp3– remained unchanged (Fig. 6D). Due to the small percentage of transferred CFSE-labeled BDC2.5NOD T cells present in the pancreas of MHV-68 infected mice, the percentage of Foxp3 expression in autoreactive CD4+ T cells could not be determined by FACS. However, analysis of the endogenous CD4+ T cell infiltrate suggested that the percentage of Foxp3 expressing cells was not increased at 7 days after i.p. MHV-68 infection (Fig. 6E). These data demonstrate that a significant reduction in the proliferation of autoreactive CD4+ T cells within the PLN of NOD mice during acute i.p. MHV-68 infection does not correlate with an expansion of CD4+ regulatory T cells.

The expression of viral and nonviral IL-10 in the islets has also been implicated in altering autoreactive CD4+ T cell activation and diabetes onset in NOD mice (22, 23). Because MHV-68 is known to induce IL-10 expression in the host, we next assessed whether local production of this cytokine might alter autoreactive CD4+ T cell activation and proliferation in the PLN and subsequent trafficking to the pancreas. IL-10 production in PLN derived CD4+ T cells was demonstrated (Fig. 7A). As this suggested that IL-10 might be involved in the decreased trafficking and activation seen in MHV-68 infected mice, we used and anti-IL-10R Ab to see if we could alter the effect of acute infection. However, use of the anti-IL-10R Ab over the course of infection and transfer of autoreactive CD4+ T cells showed that IL-10R blockade was unable to alter decreases in autoreactive CD4+ T cell trafficking, activation, and proliferation (Fig. 7B). This demonstrates that neither the induction of Ag-specific regulatory T cells, nor the local production of virally induced IL-10, are responsible for decreases in autoreactive CD4+ T cell trafficking, activation, and proliferation during acute MHV-68 infection.


Figure 7
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  		FIGURE 7. Virally induced IL-10 is not responsible for decreases in autoreactive CD4+ T cell activation and proliferation in the PLN or subsequent trafficking to the pancreas. A, Two x 107 CFSE-labeled BDC2.5NOD splenocytes were transferred to 8-wk-old female NOD mice day 7 after i.p. injection of 104 PFU MHV-68 ({blacktriangleup}) or 200 µl PBS as a control ({blacksquare}). PLN were harvested 72 h posttransfer and stained for intracellular IL-10 and an isotype control. B, NOD mice were treated with 200 µg anti-IL-10R (open symbols), an isotype control (MAC221; filled symbols), or PBS control i.p. on day 3 and 5 postinfection and directly proceeding BDC2.5NOD transfer (day 7 postinfection). Autoreactive T cell populations were transferred and tracked to the pancreas and PLN.

 
Acute virus infection affects DC endocytosis and Ag processing without affecting costimulatory molecule expression or peptide presentation

TLR ligand-mediated systemic activation can alter the ability of DCs to present new Ag to CD4+ T cells directly and to CD8+ T cells via cross-presentation. To determine whether the presence of MHV-68 within the PLN alters the ability of DCs to present autoantigen to autoreactive CD4+ T cells, we transferred labeled carboxylated yellow green microspheres to infected NOD mice and assessed the ability of DCs from the spleen and PLN to endocytose these particles. The endocytosis of labeled microspheres was significantly reduced in MHV-68 infected mice; this was comparable to the reduction in endocytosis seen following i.v. administration of LPS (Fig. 8A). Reduced endocytosis was evident in all DC subsets, including the CD11b+CD11c+ subset implicated in early self-Ag presentation and diabetes onset (24) and the CD8{alpha}+ subset implicated in self-Ag cross-presentation (25). This observation provides an explanation for the reduced activation, proliferation of autoreactive CD4+ T cells within the PLN (Fig. 5A), and the increased proportional representation of cells of a more naive phenotype (Fig. 5B).


Figure 8
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  		FIGURE 8. Acute MHV-68 infection reduces the endocytic capacity (A) and alters the phenotype (B) of DCs. A total of 3.64 x 1010 fluoresbrite YG carboxylate microspheres (0.5 µm) were transferred i.v. to 8-wk-old female NOD mice day 8 after i.p. injection of 104 PFU MHV-68 ({blacktriangledown}) or 200 µl PBS ({blacksquare}). Five micrograms of LPS was injected into uninfected mice 16 h before microsphere injection to act as a positive control for activation ({blacktriangleup}). Spleen and PLN were harvested 3 h after microsphere injection, stained, and analyzed for the endocytosis of fluorescent beads (A) and costimulatory molecule expression (B).

 
Phenotypic analysis of CD11c+ PLN DCs during acute infection showed that there was a decrease in MHC class I expression, while CD86, CD80, and MHC class II expression was unaffected (Fig. 8B). As expected, all costimulatory molecules were up-regulated following LPS administration. To assess the functional consequences of these effects on DCs, we next isolated similarly affected splenic DCs from acutely infected mice and examined their ability to present whole length protein and peptide to CD4+ T cells. Splenic DCs from acutely infected BALB/c mice were significantly impaired in their ability to present full length OVA to DO11.10 CD4+ T cells as measured by IL-2 production and proliferation (Fig. 9A). Their capacity to present peptide was unaltered during acute infection (Fig. 9B), suggesting they have the capacity for MHC class II presentation to CD4+ T cells and do not lack costimulatory molecules; however, the endocytosis, processing, and loading of full-length protein for presentation is significantly reduced during acute infection. This work suggests that MHV-68 infection can interfere with the ability of DCs to sample the environment and present autoantigen to autoreactive CD4+ T cells. As a consequence, autoreactive CD4+ T cells are poorly activated, resulting in a significant delay and reduction in diabetes onset.


Figure 9
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  		FIGURE 9. Acute MHV-68 infection reduces DC presentation of full-length protein to CD4+ T cells. A, Eight-wk-old BALB/c mice were infected with 104 PFU MHV-68 ({blacktriangleup}) or PBS as a control ({blacksquare}) and DCs positively selected with CD11c microbeads at day 10 postinfection. Their ability to present soluble OVA to 2 x 105 DO11.10 cells was assessed by IL-2 production and CFSE-labeled DO11.10 CD4+ T cell prolfieration. B, Eight-wk-old female NOD mice were injected with 104 PFU MHV-68 ({blacktriangleup}) or PBS as a control ({blacksquare}), and splenic DCs were positively selected with CD11c microbeads at day 10 postinfection. Their ability to present BDC2.5 peptide to 2 x 105 CFSE-labeled BDC2.5NOD cells was assessed by IL-2 production and BDC2.5NOD CD4+ T cell proliferation. Statistics were performed using a two-tailed non parametric t test; ***, p < 0.0001; **, ≤ 0.0086; *, ≤ 0.0249.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we demonstrated that local MHV-68 infection can reduce autoantigen presentation and delay the development of T1D in NOD mice. The acute phase of MHV-68 infection correlates with a reduction in DC endocytosis, presentation of full length Ag, autoreactive CD4+ T cell activation, and proliferation in the PLN and trafficking of autoreactive CD4+ T cells to the pancreas.

MHV-68 infection has not previously been either positively or negatively linked to diabetes development in murine models, although lytic viral infection induces the relapse of experimental autoimmune arthritis (26) and exacerbates autoimmune encephalomyelitis (27). Human gammaherpesvirus infections such as Epstein-Barr virus have been positively linked with diabetes and systemic lupus erythematosus (28). For the latter, infection can result in polyclonal B cell activation and the production autoantibodies (29). In this study, we aimed to clarify the impact of gammaherpesvirus infection on disease development in the NOD mouse model of T1D.

Interestingly, we find that MHV-68 infection negatively correlated with diabetes onset, because i.p. and i.n. infection of 8- or 9-wk-old female NOD mice resulted in a decrease and overall delay in disease onset. This observation is similar to previous reports with other viruses including the enterovirus coxsackie B4 (1) and some more pathogenic strains of coxsackie B3 (2). We find that such a delay in diabetes onset was not observed following infection of NOD mice with a MHV-68 mutant lacking the class I evasion gene MK3 (our unpublished data), nor was it seen on exposure of NOD mice to repeated injections of heat killed bacteria, previously shown to inhibit diabetes development following infection with live bacteria (17). These results suggest that diabetes delay is dependent upon active virus replication and gene expression, rather than as a result of a response to nonspecific immune activation. Our findings also add to those suggesting that virus infection can only affect diabetes development at a critical threshold of insulitic β-cell autoreactive T cell accumulation (1) by highlighting the importance of autoreactive CD4+ T cell stimulation at a 8–9 wk old time point and the important role of DCs in mediating disease progression at this time point.

Unlike a number of virus infections shown to negatively regulate diabetes, we demonstrate that MHV-68 is not particularly pancreatrophic. Therefore, we made use of established infection routes and virus mutants to question how the differing phases of viral infection impacted on T1D progression. We were also able to ask how MHV-68 infection may impact on diabetes initiation by making use of the BDC2.5NOD mouse and analyzing autoreactive CD4+ T cell trafficking and activation during MHV-68 infection.

Use of NOD mice infected with an ORF73-deficient MHV-68 mutant (FS73) demonstrated that maintenance of latency was not necessary for T1D delay. The transfer of autoreactive splenocytes into FS73-infected mice also demonstrated that maintenance of latency was not required for a reduction in the trafficking of autoreactive CD4+ T cells to the pancreas. This work was supported by data demonstrating that trafficking of autoreactive CD4+ T cells was not altered during virus persistence (day 28 postinfection) but was significantly reduced during the acute phase of infection (day 7 postinfection).

Lymphocytic choriomeningitis virus (LCMV) infection has been associated with the disruption of diabetes onset and the altered trafficking of autoaggressive CD8+ and CD4+ lymphocytes to the pancreas (3). Altered trafficking is thought to be a result of IP-10 (CXCL10) expression, which attracts autoreactive CD8+ T cells expressing the IP-10 receptor CXCR3. Blockade of the CXC ligand with anti-IP-10 has been shown to inhibit islet infiltration in LCMV-infected mice expressing the glycoprotein of LCMV under the rat insulin promoter (30). MHV-68 is known to induce IP-10 expression in the lungs of i.n. infected mice (31). The MHV-68 encoded pan-chemokine binding protein M3 (32, 33) binds human IP-10 (8) and therefore has the potential to alter chemotaxis of autoreative CD4+ T cells in a similar manner to the disruption of CCL21-induced chemotaxis of lymphocytes (34). However, we found no differences in the numbers of autoreactive CXCR3+CD4+ T cells in the PLN of MHV-68 infected NOD mice. Use of a mutant MHV-68 virus deficient in M3 demonstrated that expression of this gene is not responsible for reduced trafficking of autoreactive CD4+ T cells to the pancreas during acute phase MHV-68 infection.

We additionally analyzed the trafficking of autoreactive CFSE-labeled BDC2.5NOD CD4+ T cells to the PLN and demonstrate that this process is unaltered during virus infection. Nonetheless, these cells remain significantly more naive in terms of activation markers and proliferate significantly less in response to Ag encounter in vivo. This is in contrast to reports demonstrating that Kilham rat virus (KRV), which infects the pancreatic lymph node (not the pancreas) of BioBreeding Diabetes Resistant (BBDR) rats, selectively activates CD45RC+CD4+ Th1 cells and results in the onset of diabetes (35). This may be a reflection of the cell specificity of the virus infection as KRV transcripts were preferentially found in those activated Th1 cells. MHV-68 is not thought to infect T cells, but rather establishes latency in activated B cells, macrophages, and DCs (36). A number of factors might influence autoreactive CD4+ T cell activation and proliferation including the presence of Foxp3+ regulatory T cells (37), local production of virally induced IL-10 (22), and the systemic activation of DCs (38).

Despite BBDR rats developing T1D following KRV infection, this virus was shown to increase the percentage of CD4+CD25+ T cells in the lymph node of infected rats, although this was not due to a preferential expansion of these cells as measured by BrdU incorporation. These cells expressed high levels of CD62L and were deemed regulatory T cells as they were able to mediate suppression in vitro (39). Because CD25 is also expressed on activated T cells, we used the more definitive marker of Ag-specific regulatory T cells, Foxp3, to identify this T cell population. We find that MHV-68 infection does not alter the numbers or percentages of Foxp3 expressing BDC2.5NOD CD4+ T cells within the PLN nor does it alter the proportional representation of endogenous regulatory cells.

It has been shown that local expression of viral-IL-10 and IL-10 can prevent or promote diabetes onset in NOD mice (22, 23). MHV-68 is known to induce IL-10 production in host B and T cell populations (12) and we could demonstrate its expression in PLN CD4+ T cells following i.p. infection. IL-10 acts as an immunosuppressant during virus infection and leads to virus persistence (40); in particular, ablation of IL-10 results in decrease viral burden during MHV-68 latency as a result of increased IL-12 expression (41). The inability of anti-IL-10R Ab administered during acute infection to influence the effect of MHV-68 infection on autoreactive CD4+ T cell activation, proliferation, and trafficking suggests that IL-10 production in response to infection did not play a role in the delay and reduction of T1D.

It is known that infections can modulate DC capacity to endocytose, present, and cross-present Ag to CD4+ and CD8+ T cells, respectively (19, 38). Our analysis of DCs from the PLN and spleen of MHV-68-infected mice showed that these cells expressed a full complement of costimulatory molecules and were not typically activated in a similar way to LPS administration in vivo. Despite these phenotypic differences, DCs exposed to both stimuli had a significantly reduced capacity to endocytose labeled beads in vivo. DCs from MHV-68 infected mice were able to present peptide to Ag-specific CD4+ T cells as well as DCs from uninfected mice, but had a significantly reduced ability to present whole length soluble Ag to transgenic CD4+ T cells ex vivo. This adds to a recent publication showing that bone-marrow derived DCs infected in vitro with MHV-68 express a full array of costimulatory molecules and present viral peptides to three virus-specific hybridomas but cannot present Ag as efficiently as uninfected DCs in a MLR (42). Interestingly, this publication also demonstrates a reduction in the viability of bone-marrow derived DCs after a high multiplicity of infection. Although it is unlikely that this multiplicity of infection occurs in vivo, it is unknown how the presence of acute MHV-68 infection may affect the viability of DCs presenting self-Ag in the PLN.

We therefore propose that acute MHV-68 infection can affect DC sampling, processing, and loading of self-Ag onto MHC class II and the subsequent activation of autoreactive CD4+ T cells in the PLN of NOD mice. When occurring at a critical threshold of autoreactive CD4+ T cell accumulation within the PLN, the presence of MHV-68 infection can sway away from diabetes onset, decreasing and reducing the incidence of disease in NOD mice.


   Acknowledgments
 
We thank Dr. Chris Smith for scientific discussion and A. Jessop and the Department of Genetics at the University of Cambridge for access to the LSR. Katherine Smith was supported on the Wellsome Trust PhD programme: Infection and Immunity.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 We are grateful to the Wellcome Trust for the support of this research and provision of grant support for K.A.S. Back

2 Address correspondence and reprint requests to Professor Anne Cooke, University of Cambridge, Tennis Court Road, Cambridge, United Kingdom. E-mail address: ac{at}mole.bio.cam.ac.uk Back

3 Abbreviations used in this paper: T1D, type 1 diabetes; MHV-68, murine gammaherpesvirus-68; DC, dendritic cells; PLN, pancreatic lymph nodes; BHK-21, baby hamster kidney-21; PFU, plaque-forming units; LCMV, lymphocytic choriomeningitis virus; i.n., intranasal; KRV, Kilham rat virus. Back

Received for publication January 22, 2007. Accepted for publication September 21, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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