Sharvan Sehrawat

Immunity and immunopathology to viruses: what decides the outcome?

Many viruses infect humans and most are controlled satisfactorily by the immune system with limited damage to host tissues. Some viruses, however, do cause overt damage to the host, either in isolated cases or as a reaction that commonly occurs after infection. The outcome is influenced by properties of the infecting virus, the circumstances of infection and several factors controlled by the host. In this Review, we focus on host factors that influence the outcome of viral infection, including genetic susceptibility, the age of the host when infected, the dose and route of infection, the induction of anti-inflammatory cells and proteins, as well as the presence of concurrent infections and past exposure to cross-reactive agents.

Galectin-9/TIM-3 Interaction Regulates Virus-Specific Primary and Memory CD8+ T Cell Response

In this communication, we demonstrate that galectin (Gal)-9 acts to constrain CD8+ T cell immunity to Herpes Simplex Virus (HSV) infection. In support of this, we show that animals unable to produce Gal-9, because of gene knockout, develop acute and memory responses to HSV that are of greater magnitude and better quality than those that occur in normal infected animals. Interestingly, infusion of normal infected mice with α-lactose, the sugar that binds to the carbohydrate-binding domain of Gal-9 limiting its engagement of T cell immunoglobulin and mucin (TIM-3) receptors, also caused a more elevated and higher quality CD8+ T cell response to HSV particularly in the acute phase. Such sugar treated infected mice also had expanded populations of effector as well as memory CD8+ T cells. The increased effector T cell responses led to significantly more efficient virus control. The mechanisms responsible for the outcome of the Gal-9/TIM-3 interaction in normal infected mice involved direct inhibitory effects on TIM-3+ CD8+ T effector cells as well as the promotion of Foxp3+ regulatory T cell activity. Our results indicate that manipulating galectin signals, as can be achieved using appropriate sugars, may represent a convenient and inexpensive approach to enhance acute and memory responses to a virus infection.

Role of Tim-3/Galectin-9 Inhibitory Interaction in Viral-Induced Immunopathology: Shifting the Balance toward Regulators

Controlling chronic immunoinflammatory diseases such as lesions in the eye caused by infection with HSV represents a therapeutic challenge. Since CD4+ T cells are the primary orchestrators of lesions, targeting activated CD4+ T cell subsets and increasing the representation of cells that express regulatory function would be a logical therapeutic approach. We show that this outcome can be achieved by therapy, systemic or local, with the lectin family member galectin-9. This molecule, which is a natural product of many cell types, acts as a ligand to the inhibitory molecule TIM-3 (T cell Ig and mucin-3) that is expressed by activated but not naive T cells. We show that 50% or more of T cells in ocular lesions caused by HSV in mice express TIM-3 and that blocking signals from its natural ligand with a mAb results in more severe lesions. More importantly, the provision of additional galectin-9, either systemically or more effectively by local subconjuctival administration, diminished the severity of stromal keratitis lesions as well as the extent of corneal neovascularization. Multiple mechanisms were involved in inhibitory effects. These included apoptosis of the orchestrating effector T cells with consequent reduction of proinflammatory cytokines and an increase in the representation of two separate subtypes of regulatory cells as well as inhibitory effects on the production of molecules involved in neovascularization, an essential component of stromal keratitis pathogenesis. Our results indicate that galectin-9 therapy may represent a useful approach to control HSV-induced lesions, the most common cause of infectious blindness in the Western world.

Role of IL-17 and Th17 Cells in Herpes Simplex Virus-Induced Corneal Immunopathology

HSV-1 infection of the cornea leads to a blinding immunoinflammatory lesion of the eye termed stromal keratitis (SK). Recently, IL-17–producing CD4+ T cells (Th17 cells) were shown to play a prominent role in many autoimmune conditions, but the role of IL-17 and/or of Th17 cells in virus immunopathology is unclear. In this study, we show that, after HSV infection of the cornea, IL-17 is upregulated in a biphasic manner with an initial peak production around day 2 postinfection and a second wave starting from day 7 postinfection with a steady increase until day 21 postinfection, a time point when clinical lesions are fully evident. Further studies demonstrated that innate cells, particularly γδ T cells, were major producers of IL-17 early after HSV infection. However, during the clinical phase of SK, the predominant source of IL-17 was Th17 cells that infiltrated the cornea only after the entry of Th1 cells. By ex vivo stimulation, the half fraction of IFN-γ–producing CD4+ T cells (Th1 cells) were HSV specific, whereas very few Th17 cells responded to HSV stimulation. The delayed influx of Th17 cells in the cornea was attributed to the local chemokine and cytokine milieu. Finally, HSV infection of IL-17R knockout mice as well as IL-17 neutralization in wild-type mice showed diminished SK severity. In conclusion, our results show that IL-17 and Th17 cells contribute to the pathogenesis of SK, the most common cause of infectious blindness in the Western world.

Anti-Inflammatory Effects of FTY720 against Viral-Induced Immunopathology: Role of Drug-Induced Conversion of T Cells to Become Foxp3+ Regulators

FTY720 has been used to control inflammatory lesions, but the mechanisms by which the drug acts in vivo are poorly understood. Such mechanisms may result primarily from effects on lymphocyte and dendritic cell homing to lymphoid and inflammatory sites. We demonstrate that FTY720 may also act by causing the conversion of TCR-stimulated nonregulatory CD4+ T cells to Foxp3+CD4+ regulatory T cells and by enhancing their suppressive activity. In a model in which mice were ocularly infected with HSV, daily treatment with FTY720 resulted in significantly diminished ocular lesions. The treated animals showed increased frequencies of Foxp3+ T cells in lymphoid organs and at two inflammatory sites, namely cornea and trigeminal ganglia. In a second series of experiments, immunized DO11.10RAG2−/− animals, normally lacking endogenous Foxp3+ T cells, that were given FTY720 treatment developed high frequencies of Foxp3+ regulatory T cells in lymph nodes. Some converted cells persisted in treated animals for several weeks after drug administration was discontinued. Finally, FTY720 could effectively induce Foxp3-expressing cells from Foxp3− cells in vitro, an effect inhibited by anti-TGF-β or the proinflammatory cytokine IL-6. Accordingly, the anti-inflammatory effects of FTY720 could be mediated at least in part by its ability to cause the conversion of Ag-stimulated conventional T cells to become Foxp3+ regulators. The use of FTY720 along with Ag administration could represent a useful therapeutic means to selectively expand Ag-specific regulators, which could be valuable in many clinical situations such as allotransplants, some autoimmunities, as well as with some chronic infections.

Sortase-mediated modification of αDEC205 affords optimization of antigen presentation and immunization against a set of viral epitopes

A monoclonal antibody against the C-type lectin DEC205 (αDEC205) is an effective vehicle for delivery of antigens to dendritic cells through creation of covalent αDEC205–antigen adducts. These adducts can induce antigen-specific T-cell immune responses or tolerance. We exploit the transpeptidase activity of sortase to install modified peptides and protein-sized antigens onto the heavy chain of αDEC205, including linkers that contain nonnatural amino acids. We demonstrate stoichiometric site-specific labeling on a scale not easily achievable by genetic fusions (49 distinct fusions in this report). We conjugated a biotinylated version of a class I MHC-restricted epitope to unlabeled αDEC205 and monitored epitope generation upon binding of the adduct to dendritic cells. Our results show transfer of αDEC205 heavy chain to the cytoplasm, followed by proteasomal degradation. Introduction of a labile dipeptide linker at the N terminus of a T-cell epitope improves proteasome-dependent class I MHC-restricted peptide cross-presentation when delivered by αDEC205 in vitro and in vivo. We also conjugated αDEC205 with a linker-optimized peptide library of known CD8 T-cell epitopes from the mouse γ-herpes virus 68. Animals immunized with such conjugates displayed a 10-fold reduction in viral load.

IL-10 and Natural Regulatory T Cells: Two Independent Anti-Inflammatory Mechanisms in Herpes Simplex Virus-Induced Ocular Immunopathology

Two prominent anti-inflammatory mechanisms involved in controlling HSV-1-induced corneal immunopathology (stromal keratitis or SK) are the production of the cytokine IL-10 and the activity of natural regulatory T cells (nTregs). It is not known whether, under in vivo conditions, IL-10 and nTregs influence the corneal pathology independently or in concert. In the current study using wild-type and IL-10−/− animals, we have assessed the activity of nTregs in the absence of IL-10 both under in vitro and in vivo conditions. The IL-10−/− animals depleted of nTregs before ocular infection showed more severe SK lesions as compared with the undepleted IL-10−/− animals. In addition, nTregs purified from naive WT and IL-10−/− animals were equally able to suppress the proliferation and the cytokine production from anti-CD3-stimulated CD4+CD25− T cells in vitro. Furthermore, intracellular cytokine staining results indicated that nonregulatory cells expressing B220 and CD25 markers were the major IL-10-producing cell types in the lymphoid tissues of HSV-infected mice. In contrast, in the infected corneas, cells with the CD11b+Gr1+ phenotype along with a minor population of Foxp3−CD4+ and a few F4/80+ cells produced IL-10. Our current investigations indicate that at least two independent anti-inflammatory mechanisms are involved in limiting the corneal lesions in SK, both of which may need to be modulated to control SK therapeutically.

In Vitro-Generated Antigen-Specific CD4+ CD25+ Foxp3+ Regulatory T Cells Control the Severity of Herpes Simplex Virus-Induced Ocular Immunoinflammatory Lesions

ABSTRACT Generating and using regulatory T cells (Tregs) to modulate inflammatory disease represents a valuable approach to therapy but has not yet been applied as a means to control virus-induced immunopathological reactions. In this report, we developed a simplified technique that used unfractionated splenocytes as a precursor population and showed that stimulation under optimized conditions for 5 days with solid-phase anti-CD3 monoclonal antibody in the presence of transforming growth factor β (TGF-β) and interleukin-2 could induce up to 90% of CD4+ T cells to become Foxp3+ and able to mediate suppression in vitro. CD11c+ dendritic cells were intricately involved in the conversion process and, once modified in the presence of TGF-β, could convert Foxp3− CD4+ cells into Foxp3+ CD4+cells by producing TGF-β. The converted cells had undergone cell division, and the majority of them expressed activation markers along with surface molecules that would facilitate their migration into tissue sites. The primary reason for our study was to determine if such in vitro-converted Tregs could be used in vivo to influence the outcome of a virus-induced immunoinflammatory lesion in the eye caused by herpes simplex virus infection. We could show in three separate models of herpetic stromal keratitis that adoptive transfers of in vitro-converted Tregs effectively diminished lesion severity, especially when given in the initial phases of infection. The suppression effect in vivo appeared to be polyspecific. The protocol we have developed could provide a useful additional approach to control virus-induced inflammatory disease.

Ocular Neovascularization Caused by Herpes Simplex Virus Type 1 Infection Results from Breakdown of Binding between Vascular Endothelial Growth Factor A and Its Soluble Receptor

The normal cornea is transparent, which is essential for normal vision, and although the angiogenic factor vascular endothelial growth factor A (VEGF-A) is present in the cornea, its angiogenic activity is impeded by being bound to a soluble form of the VEGF receptor-1 (sVR-1). This report investigates the effect on the balance between VEGF-A and sVR-1 that occurs after ocular infection with HSV, which causes prominent neovascularization, an essential step in the pathogenesis of the vision-impairing lesion, stromal keratitis. We demonstrate that HSV-1 infection causes increased production of VEGF-A but reduces sVR-1 levels, resulting in an imbalance of VEGF-A and sVR-1 levels in ocular tissues. Moreover, the sVR-1 protein made was degraded by the metalloproteinase (MMP) enzymes MMP-2, -7, and -9 produced by infiltrating inflammatory cells that were principally neutrophils. Inhibition of neutrophils, inhibition of sVR-1 breakdown with the MMP inhibitor marimastat, and the provision of exogenous recombinant sVR-1 protein all resulted in reduced angiogenesis. Our results make the novel observation that ocular neovascularization resulting from HSV infection involves a change in the balance between VEGF-A and its soluble inhibitory receptor. Future therapies aimed to increase the production and activity of sVR-1 protein could benefit the management of stromal keratitis, an important cause of human blindness.

Galectin-1 Reduces the Severity of Herpes Simplex Virus-Induced Ocular Immunopathological Lesions

Stromal keratitis is a chronic immunopathological lesion of the eye caused by HSV-1 infection that can result in blindness. Because the inflammatory lesions are primarily orchestrated by Th1 cells, and to a lesser extent by Th17 cells, inhibiting their activity represents a useful form of therapy. In this study we evaluated the therapeutic potential of galectin-1 (gal-1), an endogenous lectin that in some autoimmune diseases was shown to suppress the functions of Th1 and Th17 cells. Treatment was begun at different times after ocular infection with HSV and the outcome was assessed clinically as well as for effects on various immune parameters. Treatment with recombinant gal-1 significantly diminished stromal keratitis lesion severity and the extent of corneal neovascularization. Treated mice had reduced numbers of IFN-γ– and IL-17–producing CD4+ T cells, as well as neutrophil infiltration in the cornea. Furthermore, disease severity was greater in gal-1 knockout mice compared with their wild-type counterparts. The many effects of gal-1 treatment include reduction in the production of proinflammatory cytokines and chemokines, increased production of IL-10, and inhibitory effects on molecules involved in neovascularization. To our knowledge, our findings are the first to show that gal-1 treatment represents a useful approach to control lesion severity in a virally induced immunopathological disease.