Windy Allman

Ocular and generalized myasthenia gravis induced by human acetylcholine receptor γ subunit immunization

Introduction: HLA‐DQ8 transgenic mice develop ocular myasthenia gravis (oMG), which then progresses to generalized MG (gMG) when immunized with the human acetylcholine receptor (H‐AChR) α subunit. Because the fetal AChR γ subunit is expressed in adult extraocular muscles, we anticipated that γ subunit immunization would generate an immune response to mouse AChR and induce MG in mice. Results: H‐AChR γ subunit immunization in HLA‐DQ8 mice induced an autoimmune response to mouse AChR and led to the destruction of AChR in the neuromuscular junction (NMJ) by anti‐AChR antibody and complement activation, and it triggered upregulation of AChR gene transcription. Conclusion: Our findings indicate that oMG may be induced by immunity to the AChR γ subunit. Muscle Nerve, 2012

Rapid clearance of herpes simplex virus type 2 by CD8+ T cells requires high level expression of effector T cell functions

CD8(+) T cells are important for resolution of HSV-2 lesions from the female genital epithelium. It is uncertain whether optimal clearance of viruses such as HSV-2 that cause a limited, non-systemic infection solely requires expression of effector functions by infiltrating CD8(+) T lymphocytes, or if the clearance rate is reflective of the expression level of critical effector functions. To address this, CD8(+) T cells from normal OT-I mice or OT-I mice deficient in IFNγ (IFNγ(-/-)) or the IFNγ receptor (IFNγR(-/-)) were activated in vitro in the presence of IFNγ or IL-4 to generate a series of effector populations (Tc1 and Tc2-like respectively) that secreted different levels of IFNγ and expressed different levels of HSV-specific cytolytic function. Compared with Tc1 cells, Tc2-like cells produced the type 2 cytokines IL-4 and IL-5, exhibited decreased IFNγ secretion, diminished proliferation in vitro, and decreased antigen-specific cytolysis in vivo. Clearance of an ovalbumin-expressing HSV-2 strain (HSV-2 tk(-) OVA) by adoptively transferred Tc2-like cells was delayed relative to Tc1 cell recipients. Because donor Tc2-like cells proliferated in vivo and infiltrated the infected genital epithelium similar to Tc1 cells, the diminished virus clearance by Tc2-like effector cells correlated with reduced expression of critical effector functions. Together, these results suggest that high level expression of protective T cell functions by effector T cells is necessary for optimal clearance of HSV-2 from the genital epithelium. These results have important implications for vaccines designed to elicit CD8(+) T cells against viruses such as HSV-2 that infect the genital tract.

Characterization of peripheral blood acetylcholine receptor-binding B cells in experimental myasthenia gravis.

In myasthenia gravis (MG), the neuromuscular transmission is impaired by antibodies (Abs) specific for muscle acetylcholine receptor (AChR). Anti-AChR Abs can be detected in the serum of MG patients, although their levels do not correlate with disease severity. In this study, we developed a flow cytometric assay for the detection of peripheral blood AChR-specific B cells to characterize B cell phenotypes associated with experimental autoimmune myasthenia gravis (EAMG). Alexa-conjugated AChR was used as a probe for AChR-specific B cells (B220+Ig+). Mice with EAMG had significantly elevated frequencies of AChR-specific IgG2+ and IgM+ B cells. While the frequencies of IgG2+ B cells and plasma anti-AChR IgG2 levels significantly correlated with the clinical grades of EAMG, the frequencies of IgM+ B cells and plasma anti-AChR IgM levels did not. These results indicate that the frequency of AChR-specific and IgG1+ (mouse IgG2 equivalent) peripheral blood B cells and anti-AChR IgG1 levels could be potential biomarkers for MG disease severity.

C5a is not involved in experimental autoimmune myasthenia gravis pathogenesis

C5 deficient mice are highly resistant to experimental autoimmune myasthenia gravis (EAMG) despite intact immune response to acetylcholine receptor (AChR), validating the pivotal role played by membrane attack complex (MAC, C5b-9) in neuromuscular junction destruction. To distinguish the significance of C5a from that of C5b in EAMG pathogenesis, C5a receptor (C5aR) knockout (KO) and wild-type (WT) mice were immunized with AChR to induce pathogenic anti-AChR antibodies. In contrast with C5 deficient mice, C5aR KO mice were equally susceptible to EAMG as WT mice and exhibited comparable antibody and lymphocyte proliferation response to AChR implicating that C5a is not involved in EAMG development.

Mannose-binding lectin pathway is not involved in myasthenia gravis pathogenesis

Classical complement pathway factor, C4 is required for experimental autoimmune myasthenia gravis (EAMG) pathogenesis. C4 is also a central component of the mannose binding lectin (MBL) pathway suggesting that this pathway might also be involved in MG pathogenesis. However, MBL gene deficient mice displayed intact anti-acetylcholine receptor (AChR)-immune response and neuromuscular junction (NMJ) IgG and complement accumulation following AChR-immunization. Moreover, no significant difference was observed between the serum MBL levels of 77 anti-AChR antibody positive generalized MG patients and 105 healthy controls. Therefore, MBL pathway does not play a role in EAMG/MG pathogenesis.

The increased expression of CD21 on AchR specified B cells in patients with myasthenia gravis

CD21, a major complement receptor expressed on B cells, is associated with autoimmune disorders. In the present study, we investigated the role of CD21 in pathogenesis of myasthenia gravis (MG) in relationship to anti-acetylcholine receptor (AchR) IgG (anti-AchR IgG) secretion. We detected increased surface expression of CD21 on AchR specified B cells as well as decreased surface expression of CD21 on total B cells in peripheral blood of patients with generalized MG (gMG). In addition, the serum concentrations of soluble secreted CD21 (sCD21) were decreased in patients with gMG. We also found that the level of CD21(+) AchR specified B cells correlated positively with serum anti-AchR IgG level, while the serum concentration of soluble CD21 correlated negatively with serum anti-AchR IgG level. Our data suggests that CD21 might facilitate its function on AchR specified B cell activation, resulting in the secretion of anti-AchR IgG.

Novel animal models of acetylcholine receptor antibody–related myasthenia gravis

Experimental autoimmune myasthenia gravis (EAMG) in mice has been used to unravel the pathogenic mechanisms and to be used as a preclinical model of myasthenia gravis (MG). Induction of predominantly ocular EAMG in HLA‐DQ8 transgenic mice immunized with acetylcholine receptor (AChR) subunits demonstrated the importance of nonconformationally expressed AChR subunits in extraocular muscle involvement. Wild‐type (WT) and CD4+ T cell knockout (KO) C57BL/6 mice developed EAMG upon immunization with AChR in incomplete Freunds adjuvant plus lipopolysaccharide. AChR‐specific IgG2+ B cell frequencies, estimated by Alexa‐conjugated AChR, and AChR‐reactive IgG2b levels significantly correlated with the clinical grades of EAMG in WT mice. CD4+ T cell–deficient EAMG mice exhibited AChR antibodies mainly of the IgG2b isotype, emphasizing T helper–independent B cell activation pathways in EAMG induction. These novel EAMG models have suggested that diverse immunopathological mechanisms might contribute to EAMG or MG pathogenesis.

Inhibitory IgG receptor FcγRIIB fails to inhibit experimental autoimmune myasthenia gravis pathogenesis

Deficiency of the inhibitory FcgammaRIIB renders mice susceptible to autoimmune disorders characterized with cellular infiltration of target tissue. To analyze the role of FcgammaRIIB in an antibody-mediated autoimmune disease, experimental autoimmune myasthenia gravis (EAMG), FcgammaRIIB knockout (KO) and wild-type mice were immunized with acetylcholine receptor (AChR). In contrast with previous reports, FcgammaRIIB KO mice were mildly resistant to EAMG despite preserved anti-AChR antibody production and neuromuscular junction complement deposition capacity. EAMG resistance was associated with reduced lymph node cell IL-6 and IL-10 production and increased CD4(+)CD25(+) cell ratios in lymph nodes. Our data suggest that FcgammaRIIB promotes antibody-mediated autoimmunity.

CD4 costimulation is not required in a novel LPS-enhanced model of myasthenia gravis

The potential of lipopolysaccharide (LPS) to induce antigen-specific B cell responses to acetylcholine receptor (AChR) in myasthenia gravis (MG) was evaluated in wild type (WT) and CD4-/- C57BL/6 mice. The WT mice immunized with AChR in LPS developed an MG-like disease (LPS-EAMG) similar to that induced by immunization with AChR in complete Freunds adjuvant (CFA-EAMG). CD4-/- mice were resistant to CFA-EAMG but susceptible to LPS-EAMG. LPS abrogated EAMG resistance in CD4-/- mice by increasing high-affinity anti-AChR IgG2b in sera and enhancing immune complex deposition in muscle.

Genetic deficiency of estrogen receptor alpha fails to influence experimental autoimmune myasthenia gravis pathogenesis.

Autoimmune myasthenia gravis (MG) is characterized by T cell and antibody responses to muscle nicotinic acetylcholine receptor (AChR). It is well known that MG as other autoimmune diseases is more prevalent in women than men and estrogen administration enhances experimental autoimmune MG (EAMG) severity. To determine whether estrogen influences EAMG pathogenesis through estrogen receptor alpha (ERα) activation, ERα knockout (KO) and wild-type (WT) C57BL/6 mice were immunized with AChR. ERα KO mice were equally susceptible to EAMG as WT mice and exhibited comparable antibody and immunopathological responses to AChR, suggesting a lack of involvement of ERα in EAMG pathogenesis.