Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2016 Jan 1.
Published in final edited form as: J Immunol. 2014 Nov 19;194(1):273–282. doi: 10.4049/jimmunol.1401285

IL-2/anti-IL-2 antibody complex treatment inhibits the development but not the progression of herpetic stromal keratitis

Subhash Gaddipati 1,2, Kathleen Estrada 1,3, Pushpa Rao 1,2, Andrew David Jerome 1,2, Susmit Suvas 1,2,*
PMCID: PMC4272922  NIHMSID: NIHMS639499  PMID: 25411200

Abstract

Interleukin-2 (IL-2) and anti-IL-2 antibody immune complex has recently been shown to expand the naturally occurring pool of CD4+Foxp3+ regulatory T cells (Foxp3+ Tregs). In this report, we showed that administration of IL-2/anti-IL-2 antibody immunocomplex to C57BL/6 mice, prior to corneal herpes simplex virus-1 (HSV-1) infection, significantly increased the pool of Foxp3+ Tregs when measured at early time-points post-infection. Increased numbers of Foxp3+ Tregs on day 2 and day 4 post-infection resulted in a marked reduction in the development of severe HSK. When compared to corneas from the control group, corneas from the immunocomplex-treated group showed a significant reduction in the amount of infectious virus on day 2 but not on day 4 post-infection. Reduced viral load was associated with two-fold increase in NK cell numbers in corneas from the immunocomplex-treated group of mice. Moreover, a dramatic reduction in the influx of CD4 T cells in inflamed corneas was determined on days 7 and 16 post-infection in the immunocomplex-treated group of infected mice. Immunocomplex treatment given on days 5, 6 and 7 post-infection significantly increased Foxp3+ Tregs in draining lymph nodes and in the spleen but failed to reduce the severity of HSK. In terms of the influx of CD4 T cells and granulocytes into inflamed corneas, no significant differences were noted between both groups of mice on day 16 post-infection. Our findings demonstrate that increasing Foxp3+ Tregs early but not late after infection in secondary lymphoid tissues is more efficacious in controlling the severity of HSK.

Keywords: Foxp3+ Tregs, inflammation and herpes simplex virus-1

Introduction

Herpetic stromal keratitis (HSK) is a chronic immunoinflammatory condition that develops in corneal tissue in response to recurrent herpes simplex virus-1 (HSV-1) infection of the cornea. Studies carried out in mouse models have clearly shown the involvement of both innate and adaptive immune cells in regulating the severity of HSK (1-4). Innate immune cells such as the natural killer cell (NK), dendritic cell (DC) and inflammatory monocyte (IM) have been shown to reduce the viral load in HSV-1 infected corneas (5, 6). In addition, recent studies suggest the involvement of CD8 T cells in clearing HSV-1 from corneas at late time-points post-infection (7). Most importantly, the damage to corneal tissue is caused largely because of the presence of overwhelming numbers of neutrophils and CD4 T cells in inflamed cornea (1, 2). Therefore, strategies to promote viral clearance, reduce the numbers of neutrophils, and reduce the numbers of CD4 T cells in inflamed corneas could be highly effective in controlling virus-induced corneal inflammation.

Earlier, we have shown that depletion of the naturally occurring pool of CD25+ Foxp3+ regulatory T cells (Foxp3+ Tregs) enhances the infiltration of CD4 T cells into the inflamed cornea resulting in the development of severe HSK (8). Adoptive transfer of ex-vivo generated antigen specific Foxp3+ Tregs has also been shown to control the severity of HSV-1 induced immunoinflammatory reactions in inflamed corneas (9). In addition, increasing the ratio of Foxp3+ Tregs to T effectors has been shown to reduce the severity of HSK (10). CD25+Foxp3+ Tregs have also been reported in rabbit conjunctiva, where they suppress virus specific effector CD4 and CD8 T cells during ocular HSV-1 infection (11). Together, these studies show the role of polyclonal and antigen specific Foxp3+ Tregs in controlling HSK severity in animal models.

Recently, administration of IL-2/anti-IL-2 JES6-1 monoclonal antibody immunocomplex (IL-2/JES6-1 immunocomplex) is reported to dramatically increase the numbers of naturally occurring pool of Foxp3+ Tregs (12). This approach has been used to ameliorate many inflammatory conditions in animal models (13-15). In this study, IL-2/JES6-1 immunocomplex was systemically administered prior to or late after the corneal HSV-1 infection in order to expand the pool of naturally occurring Foxp3+ Tregs in C57BL/6 mice. Our results showed that expanding Foxp3+ Tregs early after HSV-1 infection significantly reduced the development of severe HSK. This was associated with a marked increase in the influx of NK cells into inflamed corneas and a reduced viral load on day 2 post-infection. However, the depletion of NK cells did not affect the reduced viral load noted in immunocomplex-treated mice. Most importantly, a dramatic reduction in the numbers of CD4 T cells in inflamed corneas of the IL-2/JES6-1 immunocomplex treated group of mice was noted on days 7 and 16 post-infection. A significant reduction in the numbers of HSV-1 specific interferon gamma producing CD4 T cells was determined in the draining lymph nodes and in the spleen of the IL-2/JES6-1 immunocomplex treated group when compared with the control group of infected mice. On the other hand, expanding Foxp3+ Tregs at late time-points after infection did not significantly reduce the severity of HSK. No significant differences in the numbers of CD4 T cells and neutrophils were determined in the inflamed corneas from both groups of mice when measured on day 16 post-infection. Our findings demonstrate that increasing the pool of naturally occurring Foxp3+ Tregs in secondary lymphoid tissues early but not late after corneal HSV-1 infection is effective in controlling the severity of HSK.

Methods

Mice

Eight to twelve weeks old female C57BL/6 (B6) mice were procured from The Jackson Laboratory (Bar Harbor, ME) and were housed in Association for Assessment and Accreditation of Laboratory Animal Care (AALAC)-approved animal facility at Oakland University. Special instructions were given to Jackson labs to ensure that mice had no corneal opacity upon arrival. Animals were sex and age-matched for all experiments. All manipulations were performed in a type II biosafety cabinet. All experimental procedures were in complete agreement with the Association for Research in Vision and Ophthalmology resolution on the use of animals in research. In addition, all procedures were carried out in accordance with the rules and regulations of The Institutional Animal Care and Use Committee (IACUC) of the Oakland University.

Virus

HSV-1 RE used in the current study was obtained from Dr. Robert Hendricks lab at University of Pittsburgh School of Medicine, Pittsburgh, PA. The virus was propagated on monolayer of Vero cells (American Type Culture Collection, Manassas, VA; CCL81) as described previously. Pellets of infected Vero cells were suspended in 2 ml of PBS followed by three cycles of rapid freeze thaw with liquid nitrogen. Virus purified from the supernatant was titrated on Vero cells and stored in aliquots at −80°C until used.

Corneal HSV-1 infection

To carry out ocular HSV-1 infection, mice were first anesthetized by intra-peritoneal injection of avertin (Sigma-Aldrich, St. Louis, MO) at a dose of 250 mg/kg in 0.25ml PBS. The cornea was scarified with trephine (Fine Science Tools, Foster city, CA) while twisting three to four times over the corneal surface. The 1×106 or 1×105 plaques forming unit (p.f.u) of HSV-1 virus was then applied to the eye in 3μl of phosphate buffered solution (PBS) followed by gentle massage of the eyelids.

Clinical Scoring of HSK

The eyes were examined on days 10 and 16 post-infection, while using a hand-held slit lamp biomicroscope (Kowa, Nagoya, Japan), to determine the extent of the corneal opacity and angiogenesis. A standard scale for opacity, ranging from 0-5, was used as described earlier (16) with brief modification. Briefly, the score of 0 was given to normal cornea; 0.5, localized corneal haze in the paracentral or central cornea; 1, mild corneal haze; 1.5 mild corneal haze with epithelial defect; 2, moderate opacity with iris visible; 2.5, moderate opacity with epithelial defect; 3, severe opacity with iris not visible; 3.5 severe opacity with iris not visible and epithelial defect; 4, severe opacity with corneal ulcer; 4.5, severe opacity, corneal ulcer and central epithelial defect; and 5, corneal rupture. The neovascularization (NV) of the cornea was determined by measuring the centripetal growth of newly formed blood vessels in each quadrant of the cornea as described earlier (17). Briefly, the length of blood vessels in each quadrant is graded between 0 (no NV) and 4 (NV reached the central region of the cornea) in increments of approximately 1/4th of the corneal radius. The final score from four quadrants is summed to derive the NV index (total range, 0-16) for each eye at a given time point.

IL-2/anti-IL-2 immunocomplex preparation and in vivo injection

IL-2/anti-IL-2 monoclonal antibody (JES6-1) immunocomplex was prepared as described earlier (12). Briefly, the IL-2 immunocomplex was prepared in the ratio of 1μg IL-2 (Peprotech) to 5μg anti-IL-2 JES6-1 (Bio X cell) per mouse and added to 20 μl of PBS. The mixture was then incubated in 37°C water bath for 30 minutes. At the end of incubation period, a volume of 1× PBS was added to the mixture so that each mouse received 200 μl of intraperitoneal injection. The injections were given for three days either on −3, −2, −1 days prior to ocular HSV-1 infection or on +5, +6, +7 day post-ocular infection.

Viral titration

HSV-1 load in infected corneas of PBS and IL-2/anti-IL-2 immunocomplex treated groups of mice were determined by eye-swab method. Briefly, the infected corneas were swabbed with sterile Q-tips soaked in serum free DMEM on day 2 and 4 post-infection. The tip of swab was removed and placed in 500μl of serum free DMEM, which was then stored at −80° C. Before titrating swabbed samples, the cotton tip was compressed in a 3ml syringe to release excess media. Quantification of infectious virus was performed using standard Vero cell plaque assay. Cells were grown in a 12 well plate. Samples to be tested for viral load were serially diluted in serum free DMEM and added to each well. Plates were incubated at 37°C and 5% CO2 for 1 hour. Samples were then carefully removed and replaced with 1 ml of MEM/CMC overlay media. Plates were kept in incubator (37°C and 5% CO2) for 48 hours to allow the plaque formation. At the end of incubation period, overlay media was removed and 1 ml of glutaraldehyde (50 times diluted in PBS) was added in each well for 5 minutes. After removing the fixative, cells were finally stained with Coomassie blue for 1 hour at 37°C. Plaques were then counted to determine the number of plaque forming units (p.f.u) per cornea.

Corneal digestion

The infected eyes were enucleated and put into RPMI medium. Under dissecting microscope, a radial incision was made at the limbal region and the cornea was separated out from the underlying lens, iris, ciliary body and scleral tissue using curved fine forceps (Miltex surgical instruments, PA). Individual cornea was put into 250 μl of RPMI only and 20 μl of liberase TL (2.5 mg/ml) was added followed by incubation at 37°C for 45 minutes on disruptor genie. At the end of incubation period, the samples were triturated using 3 ml syringe plunger and passed through 70 μm cell strainer. Finally, the single cell suspension was washed with 5ml RPMI+10%FBS and the cells were pellet down at 315 × g for 8 minutes in a refrigerated centrifuge.

Cell surface staining

The single cell suspensions from individual corneas were washed with FACS buffer (PBS+2%FBS+0.1% sodium azide) followed by blocking of the Fc receptors with anti-mouse CD16/32 antibody on ice. The following antibodies were used for cell surface staining: Percp-cy5.5 conjugated-anti-CD11b (M1/70) and anti-CD4 (RM4-5) antibodies, PE-conjugated anti-NK1.1 (PK136), APC conjugated anti-CD3 (145-2C11), FITC conjugated anti-Ly6G (1A8) and anti-Gr1 (RB6-8C5), FITC conjugated anti CD49b (DX5), PE-cy7 conjugated anti-CD45 (30-F11) antibodies. All antibodies except anti-Foxp3 antibody (e-biosciences) were bought from BD biosciences, San Diego, CA. At the end of cell surface staining, samples were either immediately acquired on a FACS canto II flow cytometer or incubated overnight in perm-fix buffer as per the manufacturer (e-biosciences Foxp3 staining kit) instruction for Foxp3 staining. Data was analyzed using FlowJo software.

Intracellular cytokine staining

Viral stimulation

HSV-1 infected C57BL/6 mice from PBS and IL-2 immunocomplex treated groups were euthanized on day 7 post-infection. At the same time a group of uninfected C57BL/6 mice was also euthanized. Spleen and draining lymph nodes (DLNs) were isolated and collected in RPMI+10%FBS medium. Single cell suspension was prepared by triturating the lymphoid tissues with 3 ml syringe plungers followed by passing through a 70 μm cell strainer. Red blood cells (RBCs) were lysed with red blood cell lysis buffer (sigma) and cells were counted using a hemocytometer under an inverted phase microscope. Single cell suspensions of the splenocyte and lymph node were stimulated with 3 MOI (multiplicity of infection) of UV inactivated HSV-1. Virus was inactivated at 120,000 μJ/cm2 for 1 min using XL-1000 UV cross linker (Spectronics Corporation). Cells were stimulated in a 96 well u-bottom plate for 16h at 37°C in CO2 incubator. During last four hours of incubation, 1 μl Brefeldin A (BD Golgiplug) was added to each well. At the end of incubation period, cell surface staining was carried out as described above followed by permeabilization of cells with Cytofix/Cytoperm (BD biosciences). PE-conjugated anti-IFN-γ (XMG1.2) antibody was used to stain IFN-γ expressing CD4 T cells. Cells were washed in perm wash (BD Biosciences) buffer with final washing given in FACS buffer. Samples were then acquired on FACS canto II flow cytometer and the data was analyzed using FlowJo software.

PMA and Ionomycin stimulation

Single cell suspensions of spleen and corneas from HSV-1 infected groups of mice were prepared on day 16 post-infection. Cells were plated at 1.5 × 106 cells/well in a 96 well plate. Cells were stimulated with phorbol myristate acetate (100 ng/mL) and Ionomycin (500 ng/mL) in RPMI + 10% FBS. After two hours of incubation at 37°C in CO2 incubator, 1 μl Brefeldin A (BD Golgiplug) was added to each well for last 4 hours of incubation followed by intracellular cytokine staining as described above.

In vivo depletion of NK cells and Foxp3+ Tregs

Uninfected C57BL/6 mice were given intra-peritoneal injection of 250 ug of rat anti-mouse NK1.1 antibody (PK-136 from BioXcell, NH, USA) on day 4 and day 1 before ocular HSV-1 infection. The control group of mice received same dose of Rat IgG. Both groups of mice were given IL-2/JES6-1 treatment as described above. Depletion of NK cells was confirmed with anti-CD49 (DX5) antibody staining carried out on the splenocytes from both groups of mice at day 2 post-infection. Similarly, in another set of experiment, C57BL/6 mice received intra-peritoneal administration of GK1.5 (250 ug/ mouse) on days 4 and 1 before corneal HSV-1 infection. The control group of mice received Rat IgG. Both groups were administered with IL-2/JES6-1 immunocomplex prior to infection. Another group of mice received only PBS prior to infection. Mice from all three groups were euthanized on day 2 post-infection and corneas were processed for flow cytometery.

Statistical Analysis

Statistical analysis was carried out using Graph Pad Prism software (San Diego, CA). All p values were calculated using an unpaired, two-tailed Student’s t test. p < 0.05 was considered statistically significant. Results are displayed as the mean ± SEM.

Results

IL-2/JES6-1 immunocomplex treatment increases the number of Foxp3+ Tregs in secondary lymphoid tissue at early time-points post corneal HSV-1 infection

Systemic administration of IL-2/JES6-1 immunocomplex is known to increase the pool of naturally occurring Foxp3+ Tregs in lymphoid tissues of uninfected mice (12). To determine whether the same approach can also increase the number of Foxp3+ Tregs in HSV-1 infected mice, a group of C57BL/6 mice was given three intra-peritoneal injections of IL-2/JES6-1 immunocomplex as described in the methods beginning, three days prior to the corneal HSV-1 infection (Figure 1A). The control group of infected mice was given intra-peritoneal injections of PBS only. Mice were euthanized on day 2 and day 4 post-infection and the numbers of Foxp3 expressing CD4 T cells were determined in spleen (Figure 1A). As shown in Figure 1B, on day 2 post-infection, there was an approximate four-fold increase in the numbers of Foxp3+ Tregs in the spleen of the immunocomplex-treated group versus the PBS treated control group of infected mice. By day 4 post-infection, the numbers of Foxp3+ Tregs were still significantly higher in the immunocomplex-treated group of mice (Figure 1B). The peak of the expansion of Foxp3+ Tregs was determined on Day 5 post IL-2/JES6-1 immunocomplex treatment or on day 2 post-corneal HSV-1 infections. Immunocomplex treatment also resulted in a significant increase in the numbers of splenic NK cells (Figure 1C). Although a moderate increase in the numbers of inflammatory monocytes (CD11b+Ly6Chi) was determined in immunocomplex-treated mice, the differences were not statistically significant (Figure 1D). The numbers of CD8 T cells were similar between both groups of mice (Figure 1E).

Figure 1.

Figure 1

Open in a new tab

IL-2/JES6-1 treatments given prior to corneal HSV-1 infection increase the numbers of Foxp3+ Tregs at early time-points post-infection. (A) Diagram depicts the IL-2/JES6-1 treatment scheme in C57BL/6 mice. (B) Representative FACS plots denoting the frequencies of Foxp3+CD4+ T cells in spleen tissue of HSV-1 infected mice on day 2 and day 4 post-infection. The FACS plots were derived from gated CD4+ T cells. Bar diagrams demonstrate the numbers of Foxp3+ Tregs in spleen tissue of individual mouse from both groups on day 2 and day 4 post-infection. Representative FACS plots and bar diagrams denoting the frequencies and numbers of NK cells (C), inflammatory monocytes (D) and CD8 T cells (E) in the splenocytes from both groups of mice on day 2 post-infection are shown. Data were analyzed using two-tail student’s t-test (****p<0.0001, **p<0.01 and *p<0.05). Data shown are representative of two similar experiments.

Increased pool of Foxp3+ Tregs at early time-points after corneal HSV-1 infection reduces the development of severe HSK

We next determined if increased numbers of Foxp3+ Tregs in immunocomplex-treated HSV-1 infected mice affected the development of virus-induced corneal opacity and angiogenesis. As described above, one group of C57BL/6 mice received intra-peritoneal injections of IL-2/JES6-1 immunocomplex whereas the control group received PBS injections three days prior to corneal HSV-1 infection. Opacity and neovascularization of the cornea was scored using hand-held slit lamp microscope as stated in the methods section on days 10 and 16 post-infection. As is evident in Figure 2A, significantly reduced corneal opacity was noted on day 10 post-infection in the immunocomplex-treated group of mice. By day 16 post-infection, clear-cut differences in the corneal opacity were evident between the PBS treated and immunocomplex-treated groups of mice. In addition, the numbers of eyes with severe corneal opacity (score≥3) were much higher in the PBS treated group than in the immunocomplex treated group of mice (Figure 2B). Significant difference in angiogenesis was evident on day 16 but not on day 10 post-infection, when immunocomplex-treated and control groups of infected animals were compared (Figure 2C). Taken together, our results indicate that an increase in Foxp3+ Tregs at early time-points after HSV-1 infection reduce the development of severe corneal opacity and angiogenesis.

Figure 2.

Figure 2

Open in a new tab

Increased numbers of Foxp3+ Tregs in secondary lymphoid tissues at early time-points after corneal HSV-1 infection reduce the development of severe HSK. (A) Corneal opacity score of individual eyes from both groups of mice were determined by slit lamp microscope on day 10 (D10) and 16 (D16) post-infection. Each circle (open or closed) denotes an individual eye. (B) Frequencies of eyes with corneal opacity ≥3 were compared between both groups of mice on day 10 and 16 post-infection. (C) Extent of the corneal angiogenesis was determined in individual eyes (open or closed circles) from both groups of mice on above-mentioned time-points post-infection. Graphs shown were derived from two independent experiments. p values were calculated using two-tail student’s t-test (***p<0.001, **p<0.01 and *p<0.05)

IL-2/JES6-1 immunocomplex treatment regulates viral load and NK cell numbers in HSV-1 infected corneas

To determine the underlying mechanisms for significantly reduced corneal opacity and angiogenesis in the IL-2/JES6-1 immunocomplex treated group, we ascertained the viral load and the influx of innate immune cells such as neutrophils, natural killer (NK) cells, NKT cells and inflammatory monocytes at early time-points post-infection in the infected corneal samples of the PBS treated and immunocomplex-treated groups of mice. As shown in Figure 3, when compared with the PBS treated control group, a significant reduction in the viral load was noted in the corneal swabs obtained from immunocomplex-treated mice on day 2 post-infection. No significant differences in the viral load of infected corneas were evident on day 4 post-infection (Figure 3). When looked at the influx of innate immune cells in infected corneas from both groups of mice on day 2 post-infection, a significant increase in the numbers of NK cells (Figure 4D) but no significant increase in the numbers of neutrophils (CD11b+Ly6G+) and inflammatory monocytes (CD11b+Ly6Chi) was determined in the infected corneas from immunocomplex-treated group of mice. NK cell influx in infected tissues is coordinated by chemokines such as CCL2 (18). CCL2 is also upregulated in HSV-1 infected corneas (19). Therefore, we determined the levels of CCL2 in the corneal lysates from both groups of mice on day 2 post-infection. As shown in supplementary Figure 1, no significant difference in levels of CCL2 was determined, with ELISA assay, when compared between both groups of mice. We also determined that immunocomplex treatment given in the absence of Foxp3+ Tregs was able to significantly increased the numbers of NK cells in HSV-1 infected corneas when compared to virus-infected PBS treated control group on day 2 post-infection (Figure 4E). However, the immunocomplex treatment given in the presence of Foxp3+ Tregs further augmented the numbers of NK cells in inflamed corneas of infected mice. These results suggest that Foxp3+ Tregs in immunocomplex-treated group of mice are partially accountable for increased numbers of NK cells noted in virus infected corneas on day 2 post-infection.

Figure 3.

Figure 3

Open in a new tab

IL-2/JES6-1 treatment given prior to corneal HSV-1 infection reduces viral load in infected corneas. The viral load in individual corneas from both groups of mice was measured as plaque forming units (p.f.u.) on the days 2 and 4 post-infection. Each circle represents an individual eye from HSV-1 infected mice. P values were calculated using two-tail student’s t-test (*p<0.05 significant and p>0.05 non-significant).

Figure 4.

Figure 4

Open in a new tab

IL-2/JES6-1 immunocomplex treatment increases the numbers of NK cells in HSV-1 infected corneas. Representative FACS plots and scatter plots denoting the frequencies and absolute numbers of leukocytes (A), neutrophils (B), inflammatory monocytes (C) and NK cells (D) in infected corneas from both groups of mice on day 2 post-infection are shown. (E) Representative FACS plots and the scatter plot graph denote the frequencies and absolute numbers of NK cells in HSV-1 infected corneas from different groups of mice on day 2 post-infection. (F) Representative FACS plots demonstrating NK cell staining in splenocytes from PBS treated and anti-NK1.1 antibody treated + IL-2 immunocomplex administered groups of mice on day 2 post-infection. Scatter plot graph is showing HSV-1 load in eye swabs taken from the individual corneas (open and closed circle) of mice from three different groups on day 2 post-infection. P values were calculated using two-tail student’s t-test (**p<0.01, *p<0.05 significant and p>0.05 non-significant).

Next, to determine, whether increased numbers of NK cells in infected corneas participate in reducing the viral load on day 2 post-infection, immunocomplex-treated group of mice was depleted of NK cells using anti-NK1.1 antibody as described in the methods section. Our results showed that while NK cell depleted immunocomplex-treated group of mice had an average of higher viral load than immunocomplex-treated group with NK cells, the differences were not statistically significant. When compared with PBS treated control group, NK cell depleted immunocomplex-treated group showed non-significant reduction in the viral load (Figure 4F). Together, our results suggested the partial involvement of NK cells in reducing the viral load in HSV-1 infected corneas from immunocomplex-treated group of mice on day 2 post-infection.

IL-2/JES6-1 immunocomplex treatment reduces the influx of CD4 T cells in inflamed corneas

It is well reported that CD4 T cells in HSV-1 infected corneas play a key role in orchestrating corneal tissue damage (2, 20, 21). Therefore, we next determined the influx of CD4 T cells into HSV-1 infected corneas from both groups of mice on day 7 and 16 post-infection. Individual corneas from both groups of mice were enzyme digested as described in the methods section. The single cell suspension was then stained for CD4 T cells followed by flow cytometery. As shown in Figure 5A, on day 7 post-infection, there is a dramatic reduction in the numbers of CD4 T cells in infected corneas of the immunocomplex-treated group of mice when compared with the PBS treated infected group of mice. Despite the fact that more CD4 T cells had infiltrated inflamed corneas of the immunocomplex treated group on day 16 post-infection as compared to day 7 post-infection, the numbers of CD4 T cells were still significantly lower in the corneas of the immunocomplex treated group when compared to the corneas of the PBS treated infected group of mice (Figure 5B).

Figure 5.

Figure 5

Open in a new tab

IL-2/JES6-1 treatment reduces the influx of CD4 T cells in HSV-1 infected corneas. Representative FACS plots demonstrating the frequencies of CD4 T cells in HSV-1 infected corneas from PBS and IL-2/JES6-1 treated groups of mice on day 7 (A) and day 16 (B) post-infection. Scatter plot graphs show the numbers of CD4 T cell in individual corneas (open and closed circles) from both groups of mice on the day 7 (A) and 16 (B) post-infection. Data shown are derived from two independent experiments and p values were calculated using two-tail student’s t-test (***p<0.001 and *p<0.05).

Increased pool of Foxp3+ Tregs at early time-points post-infection reduces the numbers of IFN-secreting HSV-1 specific CD4 T cells in draining lymph nodes and spleen

Earlier reports have shown that Foxp3+ Tregs inhibit the differentiation of CD4 T cells towards IFN-secreting Th1 subtype (22-24), and the latter play an important role in the development of HSK (2). Since, IL-2/JES6-1 immunocomplex treatment increases Foxp3+ Tregs in secondary lymphoid tissues at early time-points post-infection (Figure 1), we next determined whether increased pool of Foxp3+ Tregs inhibits the differentiation of virus-specific CD4 T cells towards the Th1 subtype after corneal HSV-1 infection. On day 7 post-infection, draining lymph nodes and spleens obtained from both groups of mice were homogenized to prepare single cell suspensions. As described in the methods, single cell suspensions of DLNs and spleen were stimulated with UV-inactivated HSV-1 followed by intracellular cytokine staining to determine the numbers of IFN-secreting CD4 T cells. As shown in Figure 6, the numbers of IFN-secreting virus specific CD4 T cells were significantly reduced in the DLNs and spleen of the immunocomplex-treated group of mice. Our results suggest that increasing Foxp3+ Tregs in secondary lymphoid tissues at early time-points (day 2 and day 4) after infection inhibits the differentiation of virus specific CD4 T cells towards the Th1 subtype.

Figure 6.

Figure 6

Open in a new tab

IL-2/JES6-1 treatment inhibits the numbers of HSV-1 specific Th1 subset in the draining lymph nodes (DLNs) and spleen of infected mice. Single cell suspensions of DLNs (A) and spleen (B) obtained from uninfected and HSV-1 infected groups of mice were stimulated with UV-inactivated virus on day 7 post-infection. Representative FACS plots show the frequencies of IFN-γ secreting virus specific CD4 T cells in DLNs (A) and spleen tissue (B) from uninfected and HSV-1 infected groups of mice. FACS plots were derived from the live cell gate of FSC/SSC. Scatter plots show the numbers of IFN-γ secreting virus specific CD4 T cells in DLNs (A) and spleen tissue (B) of individual mouse (open and closed circle) from PBS and IL-2/JES6-1 treated groups. Results shown are representative of two similar experiments. Two-tail student’s t-test was used to determine the statistical significance of the results (***p<0.001 and **p<0.01).

Increasing Foxp3+ Tregs in secondary lymphoid tissues during the progression of clinical disease do not reduce the severity of HSK

Next, we ascertained if increasing Foxp3+ Tregs during the onset of the clinical disease period modulated the progression of corneal opacity and angiogenesis. One group of HSV-1 infected mice received IL-2/JES6-1 immunocomplex treatment on days 5, 6 and 7 post-infection whereas the control group of infected mice was given intra-peritoneal injection of PBS. On day 9 post-infection, mice were euthanized and the numbers of Foxp3+ Tregs in DLNs, spleens and corneas from both groups were quantified by flow cytometery. Our results showed a significant increase in the numbers of Foxp3+ Tregs in both DLNs and spleens of the immunocomplex treated mice than PBS treated mice (Figure 7A and B). However, no significant difference in the numbers of Foxp3+Tregs was determined in the inflamed corneas from both groups of mice (Figure 7C). When compared the cell-surface expression of chemokine receptors on Foxp3+ Tregs in DLNs and spleens from both groups of mice, immunocomplex treatment resulted in a significant increase in the numbers of CXCR3 expressing Foxp3+ Tregs in both DLNs and spleen tissue of infected mice on day 9 post-infection (Figure 8). In another set of experiments, the corneal opacity and angiogenesis were both measured in the immunocomplex treated and PBS treated mice. As shown in Figure 9, no significant differences in corneal opacity and angiogenesis on days 10 and 16 post-infection were evident when both groups of mice were compared. Taken together, these observations suggest that increasing the numbers of FoxP3+ Tregs in secondary lymphoid tissues at late time-points after infection are not sufficient to inhibit the progression of HSK.

Figure 7.

Figure 7

Open in a new tab

IL-2/JES6-1 treatments given during the onset of clinical disease increase the numbers of Foxp3+ Tregs in DLN and spleen but not in the inflamed cornea. Representative FACS plots and scatter plot graphs demonstrate the frequencies and absolute numbers of Foxp3+Tregs in DLNs (A), spleen (B) and inflamed corneal tissue (C) from both groups of mice on day 9 post-infection. FACS plots shown were derived from gated CD4+ T cells. Scatter plots demonstrate the numbers of Foxp3+Tregs in DLNs (A), Spleen (B) and infected corneas (C) from both groups of mice. p values were calculated using two-tail student’s t-test where **p<0.01 was statistically significant while p>0.05 was non significant (ns).

Figure 8.

Figure 8

Open in a new tab

IL-2/JES6-1 treatments given during the onset of clinical disease change the cell-surface expression of chemokine receptors on FoxP3+Tregs. Representative FACS plots denote the frequencies of chemokine receptor expressing Foxp3+Tregs in DLN (A) and spleen (B) on day 9 post-infection from both groups of mice. Absolute numbers of specific chemokine receptor expressing FoxP3+Tregs in DLN (A) Spleen (B) from both groups of mice on day 9 post-infection are shown as bar graphs (mean ± SEM, n=5/group). **p<0.01 and *p<0.05 (unpaired, two-tailed Student’s t-test).

Figure 9.

Figure 9

Open in a new tab

IL-2/JES6-1 treatments given during the clinical disease period do not change the severity of HSK. (A) Corneal opacity score of individual eyes from both groups of mice were determined by slit lamp microscope on day 10 (D10) and 16 (D16) post-infection. Each circle (open or closed) denotes an individual eye. (B) Frequencies of eyes with corneal opacity score ≥3 were compared between both groups of mice on day 10 and 16 post-infection. (C) Extent of the corneal angiogenesis was determined in individual eyes (open or closed circles) from both groups of mice on above-mentioned time-points post-infection. Graphs shown were derived from two independent experiments. P values were calculated using two-tail student’s t-test where p>0.05 was non significant (ns).

Increasing Foxp3+ Tregs in secondary lymphoid tissues during the onset of clinical disease do not reduce the influx of neutrophils and CD4 T cells in inflamed cornea

We then tested if increasing Foxp3+ Tregs at late time-points after corneal HSV-1 infection affected the influx of neutrophils and CD4 T cells into inflamed corneas. IL-2/JES6-1 immunocomplex treatment was given as described above. On day 16 post-infection, eyes were enucleated from both groups of infected mice. Single cell suspensions of the corneas were then stained for CD4 T cells and granulocytes by flow cytometery. No significant differences in the numbers of CD4 T cells and CD11b+Gr1+ granulocytes were determined when corneas from both groups of mice were compared (Figure 10 A and B). Intracellular cytokine staining carried out on inflamed corneal samples and splenocytes from both groups of mice on day 16 post-infection showed a modest, though not statistically significant, increase in the average numbers of IFN-γ secreting CD4 T cells from immunocomplex-treated group on day 16 post-infection (Figure 10 C and D).

Figure 10.

Figure 10

Open in a new tab

IL-2/JES6-1 treatments given during the onset of clinical disease do not affect the influx of CD4 T cells and granulocytes in inflamed corneas. Representative FACS plots and scatter plots show the frequencies and absolute numbers of CD4 T cells (A) and Gr1+ granulocytes (B) in infected corneas from both groups of mice on day 16 post-infection. The frequencies and absolute numbers of IFN-γ secreting CD4 T cells in inflamed corneas (C) and Spleen tissue (D) are determined by intracellular cytokine staining on day 16 post-infection from both groups of mice. p values were calculated using two-tail student’s t-test where p>0.05 was considered non-significant (ns).

Discussion

Naturally occurring Foxp3+ Tregs (thymic and peripheral Tregs) play an important role in controlling tissue inflammation under infectious and non-infectious conditions (25, 26). Therefore, enhancing the numbers and functional efficacy of these natural Tregs under both homeostatic and inflammatory conditions could serve as a prophylactic and therapeutic approach respectively to control the tissue damage. In animal models, two of the most promising approaches to expand Foxp3+ Tregs in vivo involve either stimulating tumor necrosis factor receptor superfamily member 25 (TNFRSF25) with an agonistic monoclonal antibody (MAbT25) or cytokine therapy with IL-2/anti-IL-2 JES6-1 monoclonal antibody immune complex (12, 27). Both approaches are reported to ameliorate many inflammatory conditions (15, 28). MAbT25 therapy can expand both regulatory and effector T cells because proliferating and activated effector T cells also express TNFRSF25 (28, 29). In fact, TNFRSF25 agonistic antibody approach has been shown to effectively control HSK when given before or at the time of HSV-1 infection but was not effective when given 6 days after infection since it expanded pro-inflammatory effector T cells (28). On the other hand, role of IL-2/JES6-1 immunocomplex treatment in controlling HSK severity is not known.

In this report, we determined that IL-2/JES6-1 immunocomplex treatment, given prior to corneal HSV-1 infection, expanded the naturally occurring pool of Foxp3+ Tregs at early time-points after infection. IL-2/JES6-1 immunocomplex binds to CD25 molecule on Foxp3+ Tregs and triggers the polyclonal expansion of naturally occurring Foxp3+ Tregs irrespective of their antigen specificity (30). Therefore, increased numbers of Foxp3+ Tregs noted in immunocomplex treated group of mice at early time-points post-infection are expected to be polyclonal in terms of their antigen specificity. Naturally occurring polyclonal Foxp3+ Tregs are reported to coordinate early protective immunity to HSV-2 infection by promoting the migration of NK cells to infected tissue and thereby reducing the viral load in vaginal tissue (31). We also noted an increased influx of NK cells into corneas of the IL-2/JES61 immunocomplex treated mice on day 2 post-infection and thereby suspected a similar role of Foxp3+ Tregs. However, our results showed that immunocomplex treatment given even in the absence of Foxp3+ Tregs increased the numbers of NK cells in HSV-1 infected corneas suggesting Foxp3+ Treg independent mechanism in promoting the influx of NK cells into infected corneas. HSV-1 load in the corneal tissue at early time-points post-infection is controlled by type I IFN produced by corneal epithelial cells and by innate immune cell types such as inflammatory monocytes and NK cells (5, 32). Although the link between type I interferon and NK cells in controlling corneal HSV-1 infection is not clear, it is reported that in vaccinia viral infection, Type I interferon action on NK cells enhance its effector function and thereby help the NK cells in promoting the viral clearance from infected tissues (33). Similarly, interplay between NK cells and inflammatory monocytes has recently been shown to promote vascular inflammation (34). It is possible that collective interactions between type I interferon, NK cells and inflammatory monocytes help in effectively controlling HSV-1 load in cornea at early time-point post-infection. Therefore, depletion of NK cells alone did not result in a dramatic change in the viral load in immunocomplex-treated group of mice as noted in Figure 4F.

In the development of HSK, CD4 T cells play an important role in orchestrating the corneal opacity (21, 35). Studies have shown that inhibiting the migration of CD4 T cells to HSV-1 infected cornea reduces the development of severe HSK (20, 36). Foxp3+ Tregs are reported to exert their immunosuppressive activity in secondary lymphoid tissues such as lymph nodes (37-40). In lymph nodes, Foxp3+ Tregs inhibit the proliferation and differentiation of naive antigen specific T cells and also regulate the migration of effector CD4 T cells to inflamed tissue (8, 39, 41, 42). In fact, depletion of Foxp3+ Tregs in DTR-Foxp3 mice results in the activation, proliferation and migration of T cells to multiple organs suggesting the ability of Foxp3+ Tregs in regulating the influx of CD4 T cells to non-lymphoid tissues (43). Earlier, we determined that depleting CD25+Foxp3+ Tregs with anti-CD25 antibody approach enhances the influx of effector CD4 T cells in HSV-1 infected corneas and thereby enhances the severity of HSK (8). Accordingly, in this report we showed that increasing Foxp3+ Tregs in secondary lymphoid tissues at early time-point post-infection dramatically reduces the numbers of CD4 T cells in HSV-1 infected corneas. Increased numbers of Foxp3+ Tregs in draining lymph nodes at the time of priming of virus-specific CD4 T cells, possibly inhibited the activation and proliferation of the viral antigen specific T cells resulting into the development of a smaller pool of effector CD4 T cells. In lymph nodes, Foxp3+ Tregs also inhibit the differentiation of naïve CD4 T cells toward Th1 subtype as determined in both infectious and non-infectious conditions (8, 24, 31). From the results of our study, it is clear that an increased pool of Foxp3+ Tregs in immunocomplex treated mice reduces the numbers of HSV-1 specific IFN-secreting CD4 T cells (Th1 subtype). Th1 subtype in HSV-1 infected corneal tissue play a key role in the development of severe HSK (2). Therefore, reducing the numbers of HSV-1 specific Th1 cells with IL-2/JES6-1 immunocomplex treatment possibly played an important role in preventing the development of severe HSK.

In more common clinical situations, where ongoing corneal inflammation needs to be controlled, our results showed that IL-2/JES6-1 immunocomplex treatment, given during the onset of clinical disease, did not decrease the progression of severe corneal opacity and angiogenesis even though the treatment increased the numbers of Foxp3+ Tregs in secondary lymphoid tissues. Increasing Foxp3+ Tregs in lymph nodes and spleen at late time-point post-infection could not reduce the numbers of CD4 T cells and granulocytes in inflamed cornea. Moreover, there was a moderate increase in the numbers of IFN-γ secreting CD4 T cells in the immunocomplex treated group of mice as shown in Figure 10. These results suggest that the immunocomplex treatment given during clinical disease period might also potentiate the function of effector CD4 T cells by binding to CD25 receptor on these T cells. As a result, the immunocomplex treatment looses its efficacy in controlling the severity of virus induced corneal immunoinflammatory reactions. However, combining the immunocomplex treatment with an approach that neutralize the function of effector CD4 T cells could harness the beneficial effect of the treatment during clinical disease period.

In addition to exerting their immunosuppressive activity in secondary lymphoid tissues, Foxp3+ Tregs have also been shown to migrate to the site of inflammation where they regulate the function of immune cells and control inflammation-induced tissue damage (41, 44, 45). However, our results showed that systemic IL-2/JES6-1 immunocomplex treatment given during the onset of clinical disease did not significantly increase the numbers of Foxp3+ Tregs in inflamed corneas (Figure 7C). Migration of Foxp3+ Tregs to inflamed tissue is coordinated by the expression of chemokine receptors on Tregs and their respective ligands in the inflamed tissue (45). When looked at the expression profile of different chemokine receptors (CCR5, CXCR5, CXCR2, CX3CR1, CCR4, CXCR3, CXCR4 and CCR2) on Foxp3+ Tregs in HSV-1 infected mice, we determined that IL-2/JES6-1 treatment significantly increased the numbers of CXCR3+Foxp3+ Tregs in both DLNs and spleen tissue in comparison to the PBS treated group (Figure 8). CXCR3+ Foxp3+ Tregs accumulate in inflamed tissue in order to limit type I immunity (46, 47). It will be interesting to see if combining IL-2/JES6-1 treatment with an approach that pulls CXCR3+Foxp3+Tregs from the periphery into inflamed corneal tissue (expand and pull approach) during the clinical disease period will ameliorate severity of HSK. It is also possible that Foxp3+ Tregs may either lose their Foxp3 expression or become ineffective in inflamed corneal tissue as suggested in other inflammatory conditions (48, 49). Consequently, additional approaches are needed to stabilize Foxp3 expression and augment the suppressive activity of Tregs in inflamed tissue.

Based on the results shown in this study, we propose that systemic IL-2/JES6-1 treatment alone is an efficacious prophylactic, but not therapeutic, approach to control the severity of HSV-1 induced corneal opacity and angiogenesis in HSK disease. Future studies combining IL-2/JES6-1 treatment with additional approaches to pull Foxp3+ Tregs in inflamed corneas and inhibit function of effector CD4 T cells might be effective in reducing the severity of HSK in a therapeutic setting.

Supplementary Material

1

Acknowledgements

We thank the staff of Biomedical research facility (BRSF) in taking good care of our experimental mice. We also thank Dr. Ashima Vohra for critical reading of our manuscript.

Supported by National Eye Institute Grant EY022417 awarded to Dr. Suvas

References

  • 1.Thomas J, Gangappa S, Kanangat S, Rouse BT. On the essential involvement of neutrophils in the immunopathologic disease: herpetic stromal keratitis. J Immunol. 1997;158:1383–1391. [PubMed] [Google Scholar]
  • 2.Niemialtowski MG, Rouse BT. Predominance of Th1 cells in ocular tissues during herpetic stromal keratitis. J Immunol. 1992;149:3035–3039. [PubMed] [Google Scholar]
  • 3.Rowe AM, St Leger AJ, Jeon S, Dhaliwal DK, Knickelbein JE, Hendricks RL. Herpes keratitis. Prog Retin Eye Res. 2013;32:88–101. doi: 10.1016/j.preteyeres.2012.08.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Inoue Y. Immunological aspects of herpetic stromal keratitis. Semin Ophthalmol. 2008;23:221–227. doi: 10.1080/08820530802111390. [DOI] [PubMed] [Google Scholar]
  • 5.Frank GM, Buela KA, Maker DM, Harvey SA, Hendricks RL. Early responding dendritic cells direct the local NK response to control herpes simplex virus 1 infection within the cornea. J Immunol. 2012;188:1350–1359. doi: 10.4049/jimmunol.1101968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Conrady CD, Zheng M, Mandal NA, van Rooijen N, Carr DJ. IFN-alpha-driven CCL2 production recruits inflammatory monocytes to infection site in mice. Mucosal Immunol. 2013;6:45–55. doi: 10.1038/mi.2012.46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Conrady CD, Zheng M, Stone DU, Carr DJ. CD8+ T cells suppress viral replication in the cornea but contribute to VEGF-C-induced lymphatic vessel genesis. J Immunol. 2012;189:425–432. doi: 10.4049/jimmunol.1200063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Suvas S, Azkur AK, Kim BS, Kumaraguru U, Rouse BT. CD4+CD25+ regulatory T cells control the severity of viral immunoinflammatory lesions. J Immunol. 2004;172:4123–4132. doi: 10.4049/jimmunol.172.7.4123. [DOI] [PubMed] [Google Scholar]
  • 9.Sehrawat S, Suvas S, Sarangi PP, Suryawanshi A, Rouse BT. In vitro-generated antigen-specific CD4+ CD25+ Foxp3+ regulatory T cells control the severity of herpes simplex virus-induced ocular immunoinflammatory lesions. J Virol. 2008;82:6838–6851. doi: 10.1128/JVI.00697-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Veiga-Parga T, Suryawanshi A, Rouse BT. Controlling viral immuno-inflammatory lesions by modulating aryl hydrocarbon receptor signaling. PLoS Pathog. 2011;7:e1002427. doi: 10.1371/journal.ppat.1002427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Nesburn AB, Bettahi I, Dasgupta G, Chentoufi AA, Zhang X, You S, Morishige N, Wahlert AJ, Brown DJ, Jester JV, Wechsler SL, BenMohamed L. Functional Foxp3+ CD4+ CD25(Bright+) “natural” regulatory T cells are abundant in rabbit conjunctiva and suppress virus-specific CD4+ and CD8+ effector T cells during ocular herpes infection. J Virol. 2007;81:7647–7661. doi: 10.1128/JVI.00294-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Webster KE, Walters S, Kohler RE, Mrkvan T, Boyman O, Surh CD, Grey ST, Sprent J. In vivo expansion of T reg cells with IL-2-mAb complexes: induction of resistance to EAE and long-term acceptance of islet allografts without immunosuppression. J Exp Med. 2009;206:751–760. doi: 10.1084/jem.20082824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Dinh TN, Kyaw TS, Kanellakis P, To K, Tipping P, Toh BH, Bobik A, Agrotis A. Cytokine therapy with interleukin-2/anti-interleukin-2 monoclonal antibody complexes expands CD4+CD25+Foxp3+ regulatory T cells and attenuates development and progression of atherosclerosis. Circulation. 2012;126:1256–1266. doi: 10.1161/CIRCULATIONAHA.112.099044. [DOI] [PubMed] [Google Scholar]
  • 14.Wang YM, Alexander SI. IL-2/anti-IL-2 complex: a novel strategy of in vivo regulatory T cell expansion in renal injury. J Am Soc Nephrol. 2013;24:1503–1504. doi: 10.1681/ASN.2013070718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Wilson MS, Pesce JT, Ramalingam TR, Thompson RW, Cheever A, Wynn TA. Suppression of murine allergic airway disease by IL-2:anti-IL-2 monoclonal antibody-induced regulatory T cells. J Immunol. 2008;181:6942–6954. doi: 10.4049/jimmunol.181.10.6942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Banerjee K, Biswas PS, Kim B, Lee S, Rouse BT. CXCR2−/− mice show enhanced susceptibility to herpetic stromal keratitis: a role for IL-6-induced neovascularization. J Immunol. 2004;172:1237–1245. doi: 10.4049/jimmunol.172.2.1237. [DOI] [PubMed] [Google Scholar]
  • 17.Zheng M, Schwarz MA, Lee S, Kumaraguru U, Rouse BT. Control of stromal keratitis by inhibition of neovascularization. Am J Pathol. 2001;159:1021–1029. doi: 10.1016/S0002-9440(10)61777-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Morrison BE, Park SJ, Mooney JM, Mehrad B. Chemokine-mediated recruitment of NK cells is a critical host defense mechanism in invasive aspergillosis. J Clin Invest. 2003;112:1862–1870. doi: 10.1172/JCI18125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Kim B, Sarangi PP, Lee Y, Deshpande Kaistha S, Lee S, Rouse BT. Depletion of MCP-1 increases development of herpetic stromal keratitis by innate immune modulation. J Leukoc Biol. 2006;80:1405–1415. doi: 10.1189/jlb.0406295. [DOI] [PubMed] [Google Scholar]
  • 20.Lee SK, Choi BK, Kang WJ, Kim YH, Park HY, Kim KH, Kwon BS. MCP-1 derived from stromal keratocyte induces corneal infiltration of CD4+ T cells in herpetic stromal keratitis. Mol Cells. 2008;26:67–73. [PubMed] [Google Scholar]
  • 21.Keadle TL, Morris JL, Pepose JS, Stuart PM. CD4(+) and CD8(+) cells are key participants in the development of recurrent herpetic stromal keratitis in mice. Microb Pathog. 2002;32:255–262. doi: 10.1006/mpat.2002.0506. [DOI] [PubMed] [Google Scholar]
  • 22.Xu D, Liu H, Komai-Koma M, Campbell C, McSharry C, Alexander J, Liew FY. CD4+CD25+ regulatory T cells suppress differentiation and functions of Th1 and Th2 cells, Leishmania major infection, and colitis in mice. J Immunol. 2003;170:394–399. doi: 10.4049/jimmunol.170.1.394. [DOI] [PubMed] [Google Scholar]
  • 23.Taylor JJ, Mohrs M, Pearce EJ. Regulatory T cell responses develop in parallel to Th responses and control the magnitude and phenotype of the Th effector population. J Immunol. 2006;176:5839–5847. doi: 10.4049/jimmunol.176.10.5839. [DOI] [PubMed] [Google Scholar]
  • 24.Shen E, Zhao K, Wu C, Yang B. The suppressive effect of CD25+Treg cells on Th1 differentiation requires cell-cell contact partially via TGF-beta production. Cell Biol Int. 2011;35:705–712. doi: 10.1042/CBI20100528. [DOI] [PubMed] [Google Scholar]
  • 25.Belkaid Y, Rouse BT. Natural regulatory T cells in infectious disease. Nat Immunol. 2005;6:353–360. doi: 10.1038/ni1181. [DOI] [PubMed] [Google Scholar]
  • 26.Rubtsov YP, Rasmussen JP, Chi EY, Fontenot J, Castelli L, Ye X, Treuting P, Siewe L, Roers A, Henderson WR, Jr., Muller W, Rudensky AY. Regulatory T cell-derived interleukin-10 limits inflammation at environmental interfaces. Immunity. 2008;28:546–558. doi: 10.1016/j.immuni.2008.02.017. [DOI] [PubMed] [Google Scholar]
  • 27.Schreiber TH, Wolf D, Tsai MS, Chirinos J, Deyev VV, Gonzalez L, Malek TR, Levy RB, Podack ER. Therapeutic Treg expansion in mice by TNFRSF25 prevents allergic lung inflammation. J Clin Invest. 2010;120:3629–3640. doi: 10.1172/JCI42933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.PB JR, Schreiber TH, Rajasagi NK, Suryawanshi A, Mulik S, Veiga-Parga T, Niki T, Hirashima M, Podack ER, Rouse BT. TNFRSF25 agonistic antibody and galectin-9 combination therapy controls herpes simplex virus-induced immunoinflammatory lesions. J Virol. 2012;86:10606–10620. doi: 10.1128/JVI.01391-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Screaton GR, Xu XN, Olsen AL, Cowper AE, Tan R, McMichael AJ, Bell JI. LARD: a new lymphoid-specific death domain containing receptor regulated by alternative pre-mRNA splicing. Proc Natl Acad Sci U S A. 1997;94:4615–4619. doi: 10.1073/pnas.94.9.4615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Shevach EM. Application of IL-2 therapy to target T regulatory cell function. Trends Immunol. 2012;33:626–632. doi: 10.1016/j.it.2012.07.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Lund JM, Hsing L, Pham TT, Rudensky AY. Coordination of early protective immunity to viral infection by regulatory T cells. Science. 2008;320:1220–1224. doi: 10.1126/science.1155209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Conrady CD, Jones H, Zheng M, Carr DJ. A Functional Type I Interferon Pathway Drives Resistance to Cornea Herpes Simplex Virus Type 1 Infection by Recruitment of Leukocytes. J Biomed Res. 2011;25:111–119. doi: 10.1016/S1674-8301(11)60014-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Martinez J, Huang X, Yang Y. Direct action of type I IFN on NK cells is required for their activation in response to vaccinia viral infection in vivo. J Immunol. 2008;180:1592–1597. doi: 10.4049/jimmunol.180.3.1592. [DOI] [PubMed] [Google Scholar]
  • 34.Knorr M, Munzel T, Wenzel P. Interplay of NK cells and monocytes in vascular inflammation and myocardial infarction. Front Physiol. 2014;5:295. doi: 10.3389/fphys.2014.00295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Suryawanshi A, Veiga-Parga T, Rajasagi NK, Reddy PB, Sehrawat S, Sharma S, Rouse BT. Role of IL-17 and Th17 cells in herpes simplex virus-induced corneal immunopathology. J Immunol. 2011;187:1919–1930. doi: 10.4049/jimmunol.1100736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Tumpey TM, Cheng H, Cook DN, Smithies O, Oakes JE, Lausch RN. Absence of macrophage inflammatory protein-1alpha prevents the development of blinding herpes stromal keratitis. J Virol. 1998;72:3705–3710. doi: 10.1128/jvi.72.5.3705-3710.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Tadokoro CE, Shakhar G, Shen S, Ding Y, Lino AC, Maraver A, Lafaille JJ, Dustin ML. Regulatory T cells inhibit stable contacts between CD4+ T cells and dendritic cells in vivo. J Exp Med. 2006;203:505–511. doi: 10.1084/jem.20050783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Tang Q, Adams JY, Tooley AJ, Bi M, Fife BT, Serra P, Santamaria P, Locksley RM, Krummel MF, Bluestone JA. Visualizing regulatory T cell control of autoimmune responses in nonobese diabetic mice. Nat Immunol. 2006;7:83–92. doi: 10.1038/ni1289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Shafiani S, Tucker-Heard G, Kariyone A, Takatsu K, Urdahl KB. Pathogen-specific regulatory T cells delay the arrival of effector T cells in the lung during early tuberculosis. J Exp Med. 2010;207:1409–1420. doi: 10.1084/jem.20091885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Samy ET, Parker LA, Sharp CP, Tung KS. Continuous control of autoimmune disease by antigen-dependent polyclonal CD4+CD25+ regulatory T cells in the regional lymph node. J Exp Med. 2005;202:771–781. doi: 10.1084/jem.20041033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Uhlig HH, Coombes J, Mottet C, Izcue A, Thompson C, Fanger A, Tannapfel A, Fontenot JD, Ramsdell F, Powrie F. Characterization of Foxp3+CD4+CD25+ and IL-10-secreting CD4+CD25+ T cells during cure of colitis. J Immunol. 2006;177:5852–5860. doi: 10.4049/jimmunol.177.9.5852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Ring S, Schafer SC, Mahnke K, Lehr HA, Enk AH. CD4+ CD25+ regulatory T cells suppress contact hypersensitivity reactions by blocking influx of effector T cells into inflamed tissue. Eur J Immunol. 2006;36:2981–2992. doi: 10.1002/eji.200636207. [DOI] [PubMed] [Google Scholar]
  • 43.Kim JM, Rasmussen JP, Rudensky AY. Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice. Nat Immunol. 2007;8:191–197. doi: 10.1038/ni1428. [DOI] [PubMed] [Google Scholar]
  • 44.Chen Z, Herman AE, Matos M, Mathis D, Benoist C. Where CD4+CD25+ T reg cells impinge on autoimmune diabetes. J Exp Med. 2005;202:1387–1397. doi: 10.1084/jem.20051409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Ding Y, Xu J, Bromberg JS. Regulatory T cell migration during an immune response. Trends Immunol. 2012;33:174–180. doi: 10.1016/j.it.2012.01.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Koch MA, Tucker-Heard G, Perdue NR, Killebrew JR, Urdahl KB, Campbell DJ. The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation. Nat Immunol. 2009;10:595–602. doi: 10.1038/ni.1731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Redjimi N, Raffin C, Raimbaud I, Pignon P, Matsuzaki J, Odunsi K, Valmori D, Ayyoub M. CXCR3+ T regulatory cells selectively accumulate in human ovarian carcinomas to limit type I immunity. Cancer Res. 2012;72:4351–4360. doi: 10.1158/0008-5472.CAN-12-0579. [DOI] [PubMed] [Google Scholar]
  • 48.Zhou X, Bailey-Bucktrout SL, Jeker LT, Penaranda C, Martinez-Llordella M, Ashby M, Nakayama M, Rosenthal W, Bluestone JA. Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo. Nat Immunol. 2009;10:1000–1007. doi: 10.1038/ni.1774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Korn T, Reddy J, Gao W, Bettelli E, Awasthi A, Petersen TR, Backstrom BT, Sobel RA, Wucherpfennig KW, Strom TB, Oukka M, Kuchroo VK. Myelin-specific regulatory T cells accumulate in the CNS but fail to control autoimmune inflammation. Nat Med. 2007;13:423–431. doi: 10.1038/nm1564. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS639499-supplement-1.pdf (1.4MB, pdf)

RESOURCES