Rajeev K. Agarwal

Essential Role of the MyD88 Pathway, but Nonessential Roles of TLRs 2, 4, and 9, in the Adjuvant Effect Promoting Th1-Mediated Autoimmunity

Induction of tissue-specific experimental autoimmune diseases involves an obligatory adjuvant effect to trigger an innate response of a type that will drive a Th1-biased adaptive response. This is achieved by use of CFA containing mycobacteria (Mycobacterium tuberculosis), whose recognition by cells of the innate immune system depends on TLRs that signal through the adaptor molecule MyD88. We examined the role of selected components of the MyD88 pathway in promoting experimental autoimmune uveitis (EAU). Mice deficient in MyD88, TLR2, TLR4, or TLR9 were immunized with the retinal Ag interphotoreceptor retinoid-binding protein in CFA, and their EAU scores and associated immunological responses were examined. MyD88−/− mice were completely resistant to EAU and had a profound defect in Th1, but not Th2, responses to autoantigen challenge. Surprisingly, TLR2−/−, TLR4−/−, and TLR9−/− mice were fully susceptible to EAU and had unaltered adaptive responses to interphotoreceptor retinoid-binding protein. Examination of IL-1R family members, which share the common adaptor MyD88 with the TLR family, revealed that IL-1R-deficient mice, but not IL-18-deficient mice, are resistant to EAU and have profoundly reduced Th1 and Th2 responses. These data are compatible with the interpretation that TLR9, TLR4, and TLR2 signaling is either not needed, or, more likely, redundant in the adjuvant effect needed to induce EAU. In contrast, signaling through the IL-1R plays a necessary and nonredundant role in EAU and can by itself account for the lack of EAU development in MyD88 mice.

Rodent Models of Experimental Autoimmune Uveitis

The model of experimental autoimmune uveitis (EAU) in mice and in rats is described. EAU targets immunologically privileged retinal antigens and serves as a model of autoimmune uveitis in humans as well as a model for autoimmunity in a more general sense. EAU is a well-characterized, robust, and reproducible model that is easily followed and quantitated. It is inducible with synthetic peptides derived from retinal autoantigens in commonly available strains of rats and mice. The ability to induce EAU in various gene-manipulated, including HLA-transgenic, mouse strains makes the EAU model suitable for the study of basic mechanisms as well as in clinically relevant interventions.

Retroviral gene therapy with an immunoglobulin-antigen fusion construct protects from experimental autoimmune uveitis

Immunoglobulins can serve as tolerogenic carriers for antigens, and B cells can function as tolerogenic antigen-presenting cells. We used this principle to design a strategy for gene therapy of experimental autoimmune uveitis, a cell-mediated autoimmune disease model for human uveitis induced with the uveitogenic interphotoreceptor retinoid-binding protein (IRBP). A retroviral vector was constructed containing a major uveitogenic IRBP epitope in frame with mouse IgG1 heavy chain. This construct was used to transduce peripheral B cells, which were infused into syngeneic recipients. A single infusion of transduced cells, 10 days before uveitogenic challenge, protected mice from clinical disease induced with the epitope or with the native IRBP protein. Protected mice had reduced antigen-specific responses, but showed no evidence for a classic Th1/Th2 response shift or for generalized anergy. Protection was not transferable, arguing against a mechanism dependent on regulatory cells. Importantly, the treatment was protective when initiated 7 days after uveitogenic immunization or concurrently with adoptive transfer of primed uveitogenic T cells. We suggest that this form of gene therapy can induce epitope-specific protection not only in naive, but also in already primed recipients, thus providing a protocol for treatment of established autoimmunity.

Gene Transfer of Ig-Fusion Proteins Into B Cells Prevents and Treats Autoimmune Diseases

Based on the tolerogenic properties of IgG carriers and B cell Ag presentation, we developed a retrovirally mediated gene expression approach for treatment of autoimmune conditions. In this study, we show that the IgG-Ag retroviral constructs, expressing myelin basic protein (MBP) or glutamic acid decarboxylase in B cells, can be used for the treatment of murine models for multiple sclerosis and diabetes. Transduction of syngeneic B cells with MBP-IgG leads to the amelioration of ongoing experimental allergic encephalomyelitis induced by the transfer of primed cells from PL×SJL F1 mice with ongoing disease and could be effective even after symptoms appeared. This effect is specific and does not involve bystander suppression because treatment with MBP-IgG does not affect disease induced after immunization with proteolipid protein immunodominant peptide plus MBP. Interestingly, if donor B cells are derived from gld mice (Fas ligand-negative), then tolerance is not induced with a model Ag although there was no evidence for Fas ligand-mediated deletion of target T cells. In spontaneous diabetes in nonobese diabetic mice, we were able to stop the ongoing autoimmune process by treatment at 7–10 wk with glutamic acid decarboxylase-IgG retrovirally transduced B cells, or attenuate it with B cells transduced with an insulin B chain (B9–23) epitope IgG fusion protein. Furthermore, IgG fusion protein gene therapy can also protect primed recipients from Ag-induced anaphylactic shock, and thus does not cause immune deviation. These results demonstrate proof of principle for future efforts to develop this approach in a clinical setting.

T cell mechanisms in experimental autoimmune uveoretinitis: Susceptibility is a function of the cytokine response profile

This study addresses the question whether susceptibility versus resistance to experimental autoimmune uveoretinitis (EAU) is connected to a Th1-type (interferon-gamma high, interleukin-4 low), versus a Th2-type (IFN-γ low, IL-4 high) response. Primed lymph node cells of susceptible Lewis rats produced IFN-γ in response to antigen in culture and transferred EAU to syngeneic recipients, whereas lymph node cells of resistant F344 rats made no IFN-γ and did not transfer disease. Reversal of the disease pattern, by treatment of F344 rats with B. pertussis toxin and immunisation of Lewis rats with antigen in incomplete Freunds adjuvant, resulted in a parallel reversal of these response patterns. Neither strain produced significant IL-4 responses. A study of the response patterns in mice confirmed that high Th1 responders were susceptible, whereas low Th1 responders and Th2 responders were resistant. We conclude that susceptibility to EAU is connected with a Th1-dominant response, but resistance can involve either a ‘null’, F344-like response (Th1-low/Th2-low) or a Th2-dominant response.

Endogenous IRBP can be dispensable for generation of natural CD4+CD25+ regulatory T cells that protect from IRBP-induced retinal autoimmunity

Susceptibility to experimental autoimmune uveitis (EAU), a model for human uveitis induced in mice with the retinal antigen interphotoreceptor retinoid-binding protein (IRBP), is controlled by “natural” CD4+CD25+ regulatory T (T reg) cells. To examine whether endogenous expression of IRBP is necessary to generate these T reg cells, we studied responses of IRBP knockout (KO) versus wild-type (WT) mice. Unexpectedly, not only WT but also IRBP KO mice immunized with a uveitogenic regimen of IRBP in complete Freunds adjuvant (CFA) exhibited CD25+ regulatory cells that could be depleted by PC61 treatment, which suppressed development of uveitogenic effector T cells and decreased immunological responses to IRBP. These EAU-relevant T reg cells were not IRBP specific, as their activity was not present in IRBP KO mice immunized with IRBP in incomplete Freunds adjuvant (IFA), lacking mycobacteria (whereas the same mice exhibited normal T reg cell activity to retinal arrestin in IFA). We propose that mycobacterial components in CFA activate T reg cells of other specificities to inhibit generation of IRBP-specific effector T cells in a bystander fashion, indicating that effective T reg cells can be antigen nonspecific. Our data also provide the first evidence that generation of specific T reg cells to a native autoantigen in a mouse with a diverse T cell repertoire requires a cognate interaction.

Sexual dimorphism in immune response genes as a function of puberty

BackgroundAutoimmune diseases are more prevalent in females than in males, whereas males have higher mortality associated with infectious diseases. To increase our understanding of this sexual dimorphism in the immune system, we sought to identify and characterize inherent differences in immune response programs in the spleens of male and female mice before, during and after puberty.ResultsAfter the onset of puberty, female mice showed a higher expression of adaptive immune response genes, while males had a higher expression of innate immune genes. This result suggested a requirement for sex hormones. Using in vivo and in vitro assays in normal and mutant mouse strains, we found that reverse signaling through FasL was directly influenced by estrogen, with downstream consequences of increased CD8+ T cell-derived B cell help (via cytokines) and enhanced immunoglobulin production.ConclusionThese results demonstrate that sexual dimorphism in innate and adaptive immune genes is dependent on puberty. This study also revealed that estrogen influences immunoglobulin levels in post-pubertal female mice via the Fas-FasL pathway.

Activation of Invariant NKT Cells Ameliorates Experimental Ocular Autoimmunity by A Mechanism Involving Innate IFN-γ Production and Dampening of the Adaptive Th1 and Th17 Responses

Invariant NKT cells (iNKT cells) have been reported to play a role not only in innate immunity but also to regulate several models of autoimmunity. Furthermore, iNKT cells are necessary for the generation of the prototypic eye-related immune regulatory phenomenon, anterior chamber associated immune deviation (ACAID). In this study, we explore the role of iNKT cells in regulation of autoimmunity to retina, using a model of experimental autoimmune uveitis (EAU) in mice immunized with a uveitogenic regimen of the retinal Ag, interphotoreceptor retinoid-binding protein. Natural strain-specific variation in iNKT number or induced genetic deficiencies in iNKT did not alter baseline susceptibility to EAU. However, iNKT function seemed to correlate with susceptibility and its pharmacological enhancement in vivo by treatment with iNKT TCR ligands at the time of uveitogenic immunization reproducibly ameliorated disease scores. Use of different iNKT TCR ligands revealed dependence on the elicited cytokine profile. Surprisingly, superior protection against EAU was achieved with α-C-GalCer, which induces a strong IFN-γ but only a weak IL-4 production by iNKT cells, in contrast to the ligands α-GalCer (both IFN-γ and IL-4) and OCH (primarily IL-4). The protective effect of α-C-GalCer was associated with a reduction of adaptive Ag-specific IFN-γ and IL-17 production and was negated by systemic neutralization of IFN-γ. These data suggest that pharmacological activation of iNKT cells protects from EAU at least in part by a mechanism involving innate production of IFN-γ and a consequent dampening of the Th1 as well as the Th17 effector responses.

C57BL/6 Mice Genetically Deficient in IL-12/IL-23 and IFN-γ Are Susceptible to Experimental Autoimmune Myasthenia Gravis, Suggesting a Pathogenic Role of Non-Th1 Cells

Immunization with Torpedo acetylcholine receptor (TAChR) induces experimental autoimmune myasthenia gravis (EAMG) in C57BL/6 (B6) mice. EAMG development needs IL-12, which drives differentiation of Th1 cells. The role of IFN-γ, an important Th1 effector, is not clear and that of IL-17, a proinflammatory cytokine produced by Th17 cells, is unknown. In this study, we examined the effect of simultaneous absence of IL-12 and IFN-γ on EAMG susceptibility, using null mutant B6 mice for the genes of both the IL-12/IL-23 p40 subunit and IFN-γ (dKO mice). Wild-type (WT) B6 mice served as control for EAMG induction. All mice were immunized with TAChR in Freund’s adjuvant. dKO mice developed weaker anti-TAChR CD4+T cells and Ab responses than WT mice. Yet, they developed EAMG symptoms, anti-mouse acetylcholine receptor (AChR) Ab, and CD4+ T cell responses against mouse AChR sequences similar to those of WT mice. dKO and WT mice had similarly reduced AChR content in their muscles, and IgG and complement at the neuromuscular junction. Naive dKO mice had significantly fewer NK, NKT, and CD4+CD25+Foxp3+ T regulatory (Treg) cells than naive WT mice. Treg cells from TAChR-immunized dKO mice had significantly less suppressive activity in vitro than Treg cells from TAChR-immunized WT mice. In contrast, TAChR-specific CD4+ T cells from TAChR-immunized dKO and WT mice secreted comparable amounts of IL-17 after stimulation in vitro with TAChR. The susceptibility of dKO mice to EAMG may be due to reduced Treg function, in the presence of a normal function of pathogenic Th17 cells.

Disruption of CD40/CD40-Ligand Interactions in a Retinal Autoimmunity Model Results in Protection without Tolerance

We examined the role of CD40/CD40L interactions on the development of experimental autoimmune uveoretinitis (EAU), a cell-mediated, Th1-driven autoimmune disease that serves as a model for autoimmune uveitis in humans. EAU-susceptible B10.RIII mice immunized with the retinal autoantigen interphotoreceptor retinoid binding protein in CFA and treated with anti-CD40L Ab (MR1) had reduced incidence and severity of disease. Real-time PCR analysis revealed that the innate and adaptive responses of protected mice were reduced, without an obvious shift toward a Th2 cytokine profile. In contrast to some other reports, no evidence was found for regulatory cells in adoptive transfer experiments. To determine whether CD40L blockade resulted in long-term tolerance, mice protected by treatment with MR1 Ab were rechallenged for uveitis after circulating MR1 Ab levels dropped below the detection limit of ELISA. MR1-treated mice developed severe EAU and strong cellular responses to interphotoreceptor retinoid binding protein, comparable to those of control mice. These responses were higher than in mice that had not received the primary immunization concurrently with anti-CD40L treatment. We conclude that 1) CD40/CD40L interaction is required for EAU and its disruption prevents disease development; 2) CD40L blockade inhibits the innate response to immunization and reduces priming, but does not result in immune deviation; and 3) protection is dependent on persistence of anti-CD40L Abs, and long-term tolerance is not induced. Furthermore, immunological memory develops under cover of CD40L blockade causing enhanced responses upon rechallenge. Taken together, our data suggest that ongoing CD40/CD40L blockade might be required to maintain a therapeutic effect against uveitis.