Hee Kap Kang

Low-Dose Peptide Tolerance Therapy of Lupus Generates Plasmacytoid Dendritic Cells That Cause Expansion of Autoantigen-Specific Regulatory T Cells and Contraction of Inflammatory Th17 Cells

Subnanomolar doses of an unaltered, naturally occurring nucleosomal histone peptide epitope, H471–94, when injected s.c. into lupus-prone mice, markedly prolong lifespan by generating CD4+25+ and CD8+ regulatory T cells (Treg) producing TGF-β. The induced Treg cells suppress nuclear autoantigen-specific Th and B cells and block renal inflammation. Splenic dendritic cells (DC) captured the s.c.-injected H471–94 peptide rapidly and expressed a tolerogenic phenotype. The DC of the tolerized animal, especially plasmacytoid DC, produced increased amounts of TGF-β, but diminished IL-6 on stimulation via the TLR-9 pathway by nucleosome autoantigen and other ligands; and those plasmacytoid DC blocked lupus autoimmune disease by simultaneously inducing autoantigen-specific Treg and suppressing inflammatory Th17 cells that infiltrated the kidneys of untreated lupus mice. Low-dose tolerance with H471–94 was effective even though the lupus immune system is spontaneously preprimed to react to the autoepitope. Thus, H471–94 peptide tolerance therapy that preferentially targets pathogenic autoimmune cells could spare lupus patients from chronically receiving toxic agents or global immunosuppressants and maintain remission by restoring autoantigen-specific Treg cells.

Apigenin, a non-mutagenic dietary flavonoid, suppresses lupus by inhibiting autoantigen presentation for expansion of autoreactive Th1 and Th17 cells

IntroductionLupus patients need alternatives to steroids and cytotoxic drugs. We recently found that apigenin, a non-mutagenic dietary flavonoid, can sensitize recurrently activated, normal human T cells to apoptosis by inhibiting nuclear factor-kappa-B (NF-κB)-regulated Bcl-xL, cyclooxygenase 2 (COX-2), and cellular FLICE-like inhibitory protein (c-FLIP) expression. Because sustained immune activation and hyperexpression of COX-2 and c-FLIP contribute to lupus, we treated SNF1 mice that spontaneously develop human lupus-like disease with apigenin.MethodsSNF1 mice with established lupus-like disease were injected with 20 mg/kg of apigenin daily and then monitored for development of severe nephritis. Histopathologic changes in kidneys, IgG autoantibodies to nuclear autoantigens in serum and in cultures of splenocytes, along with nucleosome-specific T helper 1 (Th1) and Th17 responses, COX-2 expression, and apoptosis of lupus immune cells were analyzed after apigenin treatment.ResultsApigenin in culture suppressed responses of Th1 and Th17 cells to major lupus autoantigen (nucleosomes) up to 98% and 92%, respectively, and inhibited the ability of lupus B cells to produce IgG class-switched anti-nuclear autoantibodies helped by these Th cells in presence of nucleosomes by up to 82%. Apigenin therapy of SNF1 mice with established lupus suppressed serum levels of pathogenic autoantibodies to nuclear antigens up to 97% and markedly delayed development of severe glomerulonephritis. Apigenin downregulated COX-2 expression in lupus T cells, B cells, and antigen-presenting cells (APCs) and caused their apoptosis. Autoantigen presentation and Th17-inducing cytokine production by dendritic cells were more sensitive to the inhibitory effect of apigenin in culture, as evident at 0.3 to 3 μM, compared with concentrations (10 to 100 μM) required for inducing apoptosis.ConclusionsApigenin inhibits autoantigen-presenting and stimulatory functions of APCs necessary for the activation and expansion of autoreactive Th1 and Th17 cells and B cells in lupus. Apigenin also causes apoptosis of hyperactive lupus APCs and T and B cells, probably by inhibiting expression of NF-κB-regulated anti-apoptotic molecules, especially COX-2 and c-FLIP, which are persistently hyperexpressed by lupus immune cells. Increasing the bioavailability of dietary plant-derived COX-2 and NF-κB inhibitors, such as apigenin, could be valuable for suppressing inflammation in lupus and other Th17-mediated diseases like rheumatoid arthritis, Crohn disease, and psoriasis and in prevention of inflammation-based tumors overexpressing COX-2 (colon, breast).

Capsid-Specific Cytotoxic T Lymphocytes Recognize Three Distinct H-2Db-Restricted Regions of the BeAn Strain of Theiler's Virus and Exhibit Different Cytokine Profiles

ABSTRACT The role of virus-specific cytotoxic T lymphocytes (CTL) in Theilers murine encephalomyelitis virus (TMEV)-induced demyelinating disease, a viral model for multiple sclerosis, is not yet clear. To investigate the specificity and function of CTL generated in response to TMEV infection, we generated a panel of overlapping 20-mer peptides encompassing the entire capsid and leader protein region of the BeAn strain of TMEV. Binding of these peptides to H-2Kb and H-2Db class I molecules of resistant mice was assessed using RMA-S cells. Several peptides displayed significant binding to H-2Kb, H-2Db, or both. However, infiltrating cytotoxic T cells in the central nervous system of virus-infected mice preferentially lysed target cells pulsed with VP2111-130/121-140 or VP2121-130, a previously defined CTL epitope shared by the DA strain of TMEV and other closely related cardioviruses. In addition, at a high effector-to-target cell ratio, two additional peptides (VP2161-180 and VP3101-120) sensitized target cells for cytolysis by infiltrating T cells or splenic T cells from virus-infected mice. The minimal epitopes within these peptides were defined as VP2165-173 and VP3110-120. Based on cytokine profiles, CTL specific for these subdominant epitopes are Tc2, in contrast to CTL for the immunodominant epitope, which are of the Tc1 type. Interestingly, CTL function towards both of these subdominant epitopes is restricted by the H-2D molecule, despite the fact that these epitopes bind both H-2K and H-2D molecules. This skewing toward an H-2Db-restricted response may confer resistance to TMEV-induced demyelinating disease, which is known to be associated with the H-2D genetic locus.

Gender bias in Theiler's virus-induced demyelinating disease correlates with the level of antiviral immune responses

Multiple sclerosis is an immune-mediated disease of the CNS and shows a sex-biased distribution in which 60–75% of all cases are female. A mouse model of multiple sclerosis, Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease, also displays a gender bias. However, in the C57L/J strain of mice, males are susceptible to disease whereas females are completely resistant. In this study we determined the gender differences in the TMEV-specific immune response, which may be responsible for the gender bias in clinical disease. Our data clearly demonstrate that female C57L/J mice induce significantly higher levels of TMEV-specific neutralizing Ab as well as a stronger peripheral T cell response throughout the course of viral infection. In contrast, male mice have a higher level of TMEV-specific CD4+ and CD8+ T cell infiltration into the CNS as well as viral persistence. These results suggest that a higher level of the initial antiviral immune response in female mice may be able to effectively clear virus from the periphery and CNS and therefore prevent further disease manifestations. Male mice in contrast do not mount as effective an immune response, thereby allowing for eventual viral persistence in the CNS and continuous T cell expansion leading to clinical symptoms.

A Defect in Deletion of Nucleosome-Specific Autoimmune T Cells in Lupus-Prone Thymus: Role of Thymic Dendritic Cells

To study central tolerance to the major product of ongoing apoptosis in the thymus, we made new lines of transgenic (Tg) mice expressing TCR of a pathogenic autoantibody-inducing Th cell that was specific for nucleosomes and its histone peptide H471–94. In the lupus-prone (SWR × NZB)F1 (SNF1) thymus, introduction of the lupus TCR transgene caused no deletion, but marked down-regulation of the Tg TCR and up-regulation of endogenous TCRs. Paradoxically, autoimmune disease was suppressed in the αβTCR Tg SNF1 mice with induction of highly potent regulatory T cells in the periphery. By contrast, in the MHC-matched, normal (SWR × B10. D2)F1 (SBF1), or in the normal SWR backgrounds, marked deletion of transgenic thymocytes occurred. Thymic lymphoid cells of the normal or lupus-prone mice were equally susceptible to deletion by anti-CD3 Ab or irradiation. However, in the steady state, spontaneous presentation of naturally processed peptides related to the nucleosomal autoepitope was markedly greater by thymic dendritic cells (DC) from normal mice than that from lupus mice. Unmanipulated thymic DC of SNF1 mice expressed lesser amounts of MHC class II and costimulatory molecules than their normal counterparts. These results indicate that apoptotic nucleosomal autoepitopes are naturally processed and presented to developing thymocytes, and a relative deficiency in the natural display of nucleosomal autoepitopes by thymic DC occurs in lupus-prone SNF1 mice.

Preemptive Donor Apoptotic Cell Infusions Induce IFN-γ–Producing Myeloid-Derived Suppressor Cells for Cardiac Allograft Protection

We have previously shown that preemptive infusion of apoptotic donor splenocytes treated with the chemical cross-linker ethylcarbodiimide (ECDI-SPs) induces long-term allograft survival in full MHC-mismatched models of allogeneic islet and cardiac transplantation. The role of myeloid-derived suppressor cells (MDSCs) in the graft protection provided by ECDI-SPs is unclear. In this study, we demonstrate that infusions of ECDI-SPs increase two populations of CD11b+ cells in the spleen that phenotypically resemble monocytic-like (CD11b+Ly6Chigh) and granulocytic-like (CD11b+Gr1high) MDSCs. Both populations suppress T cell proliferation in vitro and traffic to the cardiac allografts in vivo to mediate their protection via inhibition of local CD8 T cell accumulation and potentially also via induction and homing of regulatory T cells. Importantly, repeated treatments with ECDI-SPs induce the CD11b+Gr1high cells to produce a high level of IFN-γ and to exhibit an enhanced responsiveness to IFN-γ by expressing higher levels of downstream effector molecules ido and nos2. Consequently, neutralization of IFN-γ completely abolishes the suppressive capacity of this population. We conclude that donor ECDI-SPs induce the expansion of two populations of MDSCs important for allograft protection mediated in part by intrinsic IFN-γ–dependent mechanisms. This form of preemptive donor apoptotic cell infusions has significant potential for the therapeutic manipulation of MDSCs for transplant tolerance induction.

Regulatory T Cells in Lupus

Naturally occurring, CD4+CD25+ regulatory T cells that are exported from the thymus early in life play an important role in controlling organ-specific autoimmune diseases, but they may not be critical for suppressing systemic autoimmunity in lupus. On the other hand, lupus-prone subjects appear to be deficient in generation of adaptive T-regulatory cells that can be induced by various means. We review autoantigen-specific therapeutic approaches that induce such regulatory T cells. Of particular interest are TGF-β producing CD4+CD25+ and CD8+ regulatory T cells that are induced by low dose tolerance therapy of lupus-prone mice with nucleosomal histone peptide epitopes, administered subcutaneously in subnanomolar doses. These regulatory T cells are not only efficient in suppressing autoantigen recognition and autoantibody production, but they also inhibit migration/accumulation of pathogenic autoimmune cells in the target organ, such as the kidneys of mice prone to develop lupus nephritis. We discuss why and under what conditions such therapeutic approaches would be beneficial in lupus patients and lupus-prone subjects.

The histone peptide H4 71-94 alone is more effective than a cocktail of peptide epitopes in controlling lupus: immunoregulatory mechanisms.

Tolerance therapy with nucleosomal histone peptides H471–94, H416–39, or H1′22–42 controls disease in lupus-prone SNF1 mice. It would be clinically important to determine whether a cocktail of the above epitopes would be superior. Herein, we found that compared with cocktail peptides, H471–94 monotherapy more effectively delayed nephritis onset, prolonged lifespan, diminished immunoglobulin G autoantibody levels, reduced autoantigen-specific Th1 and Th17 responses and frequency of TFH cells in spleen and the helper ability of autoimmune T cells to B cells, by inducing potent CD8 Treg cells. H471–94 therapy was superior in “tolerance spreading,” suppressing responses to other autoepitopes, nucleosomes, and ribonucleoprotein. We also developed an in vitro assay for therapeutic peptides (potentially in humans), which showed that H471–94, without exogenous transforming growth factor (TGF)-β, was efficient in inducing stable CD4+CD25+Foxp3+ T cells by decreasing interleukin 6 and increasing TGF-β production by dendritic cells that induced ALK5-dependent Smad-3 phosphorylation (TGF-β signal) in target autoimmune CD4+ T cells.

Differential Role of B Cells and IL-17 Versus IFN-γ During Early and Late Rejection of Pig Islet Xenografts in Mice

Background Xenogeneic islet transplantation is an emerging therapeutic option for diabetic patients. However, immunological tolerance to xenogeneic islets remains a challenge. Methods The current study used a pig-to-mouse discordant xenogeneic islet transplant model to examine antidonor xenogeneic immune responses during early and late rejection and to determine experimental therapeutic interventions that promote durable pig islet xenograft survival. Results We found that during early acute rejection of pig islet xenografts, the rejecting hosts exhibited a heavy graft infiltration with B220+ B cells and a robust antipig antibody production. In addition, early donor-stimulated IL-17 production, but not IFN-&ggr; production, dominated during early acute rejection. Recipient treatment with donor apoptotic 1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide-treated splenocytes significantly inhibited antidonor IL-17 response, and when combined with B cell depletion and a short course of rapamycin led to survival of pig islet xenografts beyond 100 days in approximately 65% recipients. Interestingly, treated recipients in this model experienced late rejection between 100 and 200 days posttransplant, which coincided with B cell reconstitution and an ensuing emergence of a robust antidonor IFN-&ggr;, but not IL-17, response. Conclusions These findings reveal that early and late rejection of pig islet xenografts may be dominated by different immune responses and that maintenance of long-term xenogeneic tolerance will require strategies that target the temporal sequence of antixenogeneic immune responses.

Recipient Myd88 Deficiency Promotes Spontaneous Resolution of Kidney Allograft Rejection

The myeloid differentiation protein 88 (MyD88) adapter protein is an important mediator of kidney allograft rejection, yet the precise role of MyD88 signaling in directing the host immune response toward the development of kidney allograft rejection remains unclear. Using a stringent mouse model of allogeneic kidney transplantation, we demonstrated that acute allograft rejection occurred equally in MyD88-sufficient (wild-type [WT]) and MyD88(-/-) recipients. However, MyD88 deficiency resulted in spontaneous diminution of graft infiltrating effector cells, including CD11b(-)Gr-1(+) cells and activated CD8 T cells, as well as subsequent restoration of near-normal renal graft function, leading to long-term kidney allograft acceptance. Compared with T cells from WT recipients, T cells from MyD88(-/-) recipients failed to mount a robust recall response upon donor antigen restimulation in mixed lymphocyte cultures ex vivo. Notably, exogenous IL-6 restored the proliferation rate of T cells, particularly CD8 T cells, from MyD88(-/-) recipients to the proliferation rate of cells from WT recipients. Furthermore, MyD88(-/-) T cells exhibited diminished expression of chemokine receptors, specifically CCR4 and CXCR3, and the impaired ability to accumulate in the kidney allografts despite an otherwise MyD88-sufficient environment. These results provide a mechanism linking the lack of intrinsic MyD88 signaling in T cells to the effective control of the rejection response that results in spontaneous resolution of acute rejection and long-term graft protection.