Khrishen Cunnusamy

IL-17A–Dependent CD4+CD25+ Regulatory T Cells Promote Immune Privilege of Corneal Allografts

IL-17A is a proinflammatory cytokine that has received attention for its role in the pathogenesis of several autoimmune diseases. IL-17A has also been implicated in cardiac and renal allograft rejection. Accordingly, we hypothesized that depletion of IL-17A would enhance corneal allograft survival. Instead, our results demonstrate that blocking IL-17A in a mouse model of keratoplasty accelerated the tempo and increased the incidence of allograft rejection from 50 to 90%. We describe a novel mechanism by which CD4+CD25+ regulatory T cells (Tregs) respond to IL-17A and enhance corneal allograft survival. Our findings suggest the following: 1) IL-17A is necessary for ocular immune privilege; 2) IL-17A is not required for the induction of anterior chamber-associated immune deviation; 3) Tregs require IL-17A to mediate a contact-dependent suppression; 4) corneal allograft Tregs suppress the efferent arm of the immune response and are Ag specific; 5) Tregs are not required for corneal allograft survival beyond day 30; and 6) corneal allograft-induced Treg-mediated suppression is transient. Our findings identify IL-17A as a cytokine essential for the maintenance of corneal immune privilege and establish a new paradigm whereby interplay between IL-17A and CD4+CD25+ Tregs is necessary for survival of corneal allografts.

IL-17 Promotes Immune Privilege of Corneal Allografts

Corneal allograft rejection has been described as a Th1-mediated process involving IFN-γ production. However, it has been reported that corneal allograft rejection soars in IFN-γ−/− mice or mice treated with anti–IFN-γ mAb. Th17 is a recently described IL-17A–producing Th cell population that has been linked to renal and cardiac graft rejection, which was originally thought to be Th1-mediated. We tested the hypothesis that Th17 cells mediate corneal allograft rejection in an IL-17A–dependent fashion and unexpectedly found that depletion of IL-17A increased the incidence of rejection to 90%. We demonstrate that the exacerbated rejection following depletion of IL-17A did not result from a loss of cross-regulation of Th1 cells or exaggerated delayed-type hypersensitivity responses. Instead, inhibition of the Th1 or Th17 cell lineages promoted the emergence of a Th2 cell subset that independently mediated allograft rejection. These findings demonstrate that IL-17A is not required for corneal allograft rejection and may instead contribute to the immune privilege of corneal allografts.

The Disease-Ameliorating Function of Autoregulatory CD8 T Cells Is Mediated by Targeting of Encephalitogenic CD4 T Cells in Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the CNS, and CD8 T cells are the predominant T cell population in MS lesions. Given that transfer of CNS-specific CD8 T cells results in an attenuated clinical demyelinating disease in C57BL/6 mice with immunization-induced experimental autoimmune encephalomyelitis (EAE), we investigated the cellular targets and mechanisms of autoreactive regulatory CD8 T cells. In this study we report that myelin oligodendrocyte glycoprotein peptide (MOG35–55)–induced CD8 T cells could also attenuate adoptively transferred, CD4 T cell–mediated EAE. Whereas CD8−/− mice exhibited more severe EAE associated with increased autoreactivity and inflammatory cytokine production by myelin-specific CD4 T cells, this was reversed by adoptive transfer of MOG-specific CD8 T cells. These autoregulatory CD8 T cells required in vivo MHC class Ia (KbDb) presentation. Interestingly, MOG-specific CD8 T cells could also suppress adoptively induced disease using wild-type MOG35–55-specific CD4 T cells transferred into KbDb−/− recipient mice, suggesting direct targeting of encephalitogenic CD4 T cells. In vivo trafficking analysis revealed that autoregulatory CD8 T cells are dependent on neuroinflammation for CNS infiltration, and their suppression/cytotoxicity of MOG-specific CD4 T cells is observed both in the periphery and in the CNS. These studies provide important insights into the mechanism of disease suppression mediated by autoreactive CD8 T cells in EAE.

Two different regulatory T cell populations that promote corneal allograft survival

PURPOSE To compare and contrast the T regulatory cells (Tregs) induced by anterior chamber (AC) injection of antigen with those induced by orthotopic corneal allografts. METHODS Anterior chamber-associated immune deviation (ACAID) Tregs were induced by injecting C57BL/6 spleen cells into the AC of BALB/c mice. Delayed-type hypersensitivity responses to C57BL/6 alloantigens were evaluated by a conventional ear swelling assay. Corneal allograft Tregs were induced by applying orthotopic C57BL/6 corneal allografts onto BALB/c hosts. The effects of anti-CD25, anti-CD8, anti-interferon-γ (IFN-γ), anti-IL-17A, or cyclophosphamide treatments on corneal allograft survival and ACAID were evaluated. RESULTS Administration of either anti-CD25 or anti-IFN-γ antibodies prevented the expression of ACAID and abolished the immune privilege of corneal allografts. By contrast, in vivo treatment with anti-CD8 antibody abrogated ACAID but had no effect on corneal allograft survival. Further discordance between ACAID and corneal allograft survival emerged in experiments in which the induction of allergic conjunctivitis or the administration of anti-IL-17A abolished the immune privilege of corneal allografts but had no effect on the induction or expression of ACAID. CONCLUSIONS Although orthotopic corneal allografts are strategically located for the induction of ACAID by the sloughing of corneal cells into the AC, the results reported here indicate that the Tregs induced by orthotopic corneal allografts are remarkably different from the Tregs that are induced by AC injection of alloantigen. Although both of these Treg populations promote corneal allograft survival, they display distinctly different phenotypes.

Disease exacerbation of multiple sclerosis is characterized by loss of terminally differentiated autoregulatory CD8+ T cells ☆

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS). Although its etiology remains unknown, pathogenic T cells are thought to underlie MS immune pathology. We recently showed that MS patients harbor CNS-specific CD8+ Tregs that are deficient during disease relapse. We now demonstrate that CNS-specific CD8+ Tregs were cytolytic and could eliminate pathogenic CD4+ T cells. These CD8+ Tregs were present primarily in terminally differentiated (CD27-, CD45RO-) subset and their suppression was IFNγ, perforin and granzyme B-dependent. Interestingly, MS patients with acute relapse displayed a significant loss in terminally differentiated CD8+ T cells, with a concurrent loss in expression of perforin and granzyme B. Pre-treatment of exacerbation-derived CD8+ T cells with IL-12 significantly restored suppressive capability of these cells through upregulation of granzyme B. Our studies uncover immune-suppressive mechanisms of CNS-specific CD8+ Tregs, and may contribute to design of novel immune therapies for MS.

MODULATION OF IMMUNE FUNCTION OCCURS WITHIN HOURS OF THERAPY INITIATION FOR MULTIPLE SCLEROSIS

Daily administration of FDA-approved glatiramer acetate (GA) has beneficial effects on clinical course of relapsing remitting multiple sclerosis (RRMS). Although mechanisms of GA-action have been widely investigated and partially understood, immediate immune dynamics following GA-therapy are unknown. In the present study, we characterized the immediate effects of GA on phenotype, quantity and function of immune cells in MS patients. Prominent changes in immune cells were detected within 4-12h post-first GA-injection. T-cell modulation included significantly decreased CD4/CD8 ratio, perturbed homeostasis of predominantly CD8+ T-cells, significant enhancement in CD8+ T-cell mediated suppression and inhibitory potential of induced CD4-suppressors. Changes in APC were restricted to monocytes and included reduced stimulatory capacity in MLR and significantly increased IL-10 and TNF-α production. Our study provides the first evidence that GA treatment induces rapid immunologic changes within hours of first dose. Interestingly, these responses are not only restricted to innate immune cells but also include complex modulation of T-cell functionality.

IFN-γ Blocks CD4+CD25+ Tregs and Abolishes Immune Privilege of Minor Histocompatibility Mismatched Corneal Allografts

Th1 CD4+ cells are believed to be the primary mediators of corneal allograft rejection. However, rejection of fully allogeneic C57BL/6 corneal allografts soared from 50% to 90% in both interferon‐gamma (IFN‐γ)−/− and anti‐IFN‐γ‐treated BALB/c mice. In contrast, similar deficits in IFN‐γ in BALB/c hosts enhanced immune privilege of BALB.B (minor histocompatibility [minor H] antigen‐matched, major histocompatibility complex [MHC]‐mismatched) and NZB (MHC‐matched, minor H antigen‐mismatched) corneal allografts—decreasing rejection from 80% to ∼20%. This effect of IFN‐γ was independent of CD4+ T cell lineage commitment as both anti‐IFN‐γ‐treated acceptor and rejector mice displayed a Th2 cytokine profile. The presence of IFN‐γ prevented the generation of alloantigen‐specific CD4+CD25+ T regulatory cells (Tregs) in hosts receiving either MHC only mismatched BALB.B or minor only histocompatibility (minor H)‐mismatched NZB corneal allografts. Tregs in these hosts promoted corneal allograft survival by suppressing Th2 effector cells. By contrast, IFN‐γ was necessary for the generation of CD4+CD25+ Tregs that prevented rejection of fully allogeneic C57BL/6 corneal allografts in BALB/c hosts. These findings suggest that MHC‐matching in combination with blockade of IFN‐γ holds promise as a means of enhancing corneal allograft survival.

Autoregulatory CD8 T cells depend on cognate antigen recognition and CD4/CD8 myelin determinants

Objective: To determine the antigenic determinants and specific molecular requirements for the generation of autoregulatory neuroantigen-specific CD8+ T cells in models of multiple sclerosis (MS). Methods: We have previously shown that MOG35-55-specific CD8+ T cells suppress experimental autoimmune encephalomyelitis (EAE) in the C57BL/6 model. In this study, we utilized multiple models of EAE to assess the ability to generate autoregulatory CD8+ T cells. Results: We demonstrate that alternative myelin peptides (PLP178-191) and other susceptible mouse strains (SJL) generated myelin-specific CD8+ T cells, which were fully capable of suppressing disease. The disease-ameliorating function of these cells was dependent on the specific cognate myelin antigen. Generation of these autoregulatory CD8+ T cells was not affected by thymic selection, but was dependent on the presence of both CD4+ and CD8+ T-cell epitopes in the immunizing encephalitogenic antigen. Conclusions: These studies show that the generation of autoregulatory CD8+ T cells is a more generalized, antigen-specific phenomenon across multiple neuroantigens and mouse strains, with significant implications in understanding disease regulation.

Pharmaceutical composition for treating macular degeneration (WO2012079419).

A pharmaceutical composition composed of several traditional Chinese medicines is claimed to treat age-related macular degeneration (AMD). This represents a novel and alternative therapeutic solution for wet AMD, with the potential advantage of treating both the symptoms and the underlying causes of this devastating degenerative retinal disease.