Is pathogenic humoral autoimmunity a Th1 response?

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Is pathogenic humoral autoimmunity a Th1 response? Lessons from (for) myasthenia gravis Balaji Balasa and Nora Sarvetnick Myasthenia gravis (MG) is mediated by autoantibodies to the nicotinic acetylcholine receptor

yasthenia gravis (MG) is CD41 T-cell response in tAChR-immunized (AChR). Although AChR-specific T an organ-specific automice is directed primarily against a non-self immune disease that aftAChR a146–162 peptide11, as shown in lymcells are necessary for disease phocyte proliferation assays, and does not flicts the neuromuscular development, the role of cytokines is crossreact with its murine homolog12. T-cell junction of humans1,2. The putative target not clear. Here, Balaji Balasa and autoantigen, the nicotinic acetylcholine relines or clones derived from MG patients recceptor (nAChR) of the neuromuscular juncognize immunodominant epitopes of AChR Nora Sarvetnick discuss the T tion, has been well characterized3,4. The dis(reviewed in Refs 2, 3). Nevertheless, tAChR helper 1 (Th1)/Th2 paradigm and order in MG patients is a result of loss of a146–162 peptide-specific T cells from functional nAChRs at the postsynaptic tAChR-immunized mice help AChR-specific emerging evidence that interferon g membrane and is mediated by autoantiB cells to produce autoAbs directed against (IFN-g) and interleukin 12 (IL-12) bodies (autoAbs) and complement2. Despite the mouse (m) AChR (Ref. 13). The role of are important in experimental the association of autoAbs with disease, CD81 T cells in EAMG has been controversial10,14. Although tAChR-specific T cells are ~10% of MG patients do not possess circuautoimmune MG pathogenesis. necessary for the development of disease, lating anti-AChR autoAbs, yet exhibit a very the pathogenesis of the disease stems from similar neurological disorder. It is believed that these patients might have autoAbs to unknown components of the collaborative effects of T and B cells15 and subsequent activity of the endplate5. However, the pathogenetic mechanisms of the disease autoAbs and complement16,17. are not completely understood. Because cytokines modulate the immune response to foreign and self-antigens (Ags), in this article, the contribution of cytokines to modulation of the humoral response Indirect evidence for the involvement of Th1 and Th2 to nAChRs and the subsequent development of disease is discussed. cytokines in MG Although polarized T helper 1 (Th1) responses have been implicated in the pathogenesis of organ-specific autoimmune diseases18, little is EAMG as an animal model of human MG known about the role of cytokines produced by Th-cell subsets in the To gain insight into the immunopathogenesis of MG, a model was development of autoAb-mediated EAMG. Both AChR-specific Th1 established in susceptible strains of mice6 and rats7 by immunizing and Th2 cells are documented in MG patients19, but it is not yet clear them with AChR purified from the electric organs of Torpedo californica which are essential for driving the production of pathogenic anti(tAChR) and emulsified in complete Freund’s adjuvant (CFA); the AChR Abs. Because Th2 cytokines are associated with B-cell help, model was termed experimental autoimmune myasthenia gravis (EAMG). tAChR-immunized animals make Abs against tAChR as Box 1. Characteristics of EAMG and human MG well as a pathogenic subset of Abs that crossreact with the mouse (self)-AChR (Ref. 6). EAMG shares several of the characteristic fea• Muscular weakness tures of human MG (Box 1)6. It is thought that the symptoms seen in • Flaccid paralysis (aggravated by exercise and relieved by animals suffering from MG are a result of autoAb-mediated decholinesterase inhibitors) struction or modulation of the AChR at the neuromuscular junction2. • Low amplitude miniature endplate potentials • Decrementing compound muscle action potentials • Simplification of the postsynaptic membrane at neuromusEAMG is a T-cell-dependent Ab-mediated disease cular junctions CD41 T cells specific for tAChR are required for the development of • Circulating autoantibodies to the nicotinic AChR EAMG. Mice treated with depleting anti-CD4 monoclonal Ab (Ref. Abbreviations: AChR, acetylcholine receptor; EAMG, experimental auto8) or those deficient in major histocompatibility complex (MHC) immune myasthenia gravis; MG, myasthenia gravis. 9 and CD41 T cells10 fail to develop the disease. The class II molecules

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the pathogenic effect of autoAbs in this disease favors the theory that a humoral Th2 response is responsible for the disease rather than a cell-mediated Th1 response. Consistent with this notion, AChRspecific CD41 T-cell lines displaying a Th2 cytokine profile promote in vitro differentiation of specific B cells into Ab-secreting plasma cells13. At the bulk culture level, AChR-specific interleukin 4 (IL-4)expressing CD41 T cells were detected in mice immunized with tAChR–CFA (Ref. 10). In another study, similarly immunized mice developed IgG1 autoAbs20, a hallmark of Th2-mediated immunity, among other IgG isotypes. In humans, MG has also been associated with T cells of the Th2 phenotype19,21. However, despite the popular notion that a predominant Th2 phenotype is essential for such disease, recent evidence suggests that CD41 T cells involved in MG might also include a Th1 subset. Thus, high levels of AChR-reactive interferon g (IFN-g)-secreting cells are present in the peripheral blood of MG patients19,22. Moreover, specific CD41 T cells from mice immunized with tAChR produce IFN-g and also IL-2 (Refs 23, 24). The immune sera from these mice also contain specific IgG2a autoAbs among other IgG isotypes. Furthermore, EAMG induced in Lewis rats with AChR in CFA is associated with raised levels of cells in lymphoid organs producing and secreting IFN-g in response to AChR (Ref. 25). There are experimental data, as well as theoretical considerations, that support a pathogenic role for Th1 cells in human MG (Refs 22, 26–28). Th1 cells from MG patients recognize the human AChR-a chain peptides 48–67, 101–137, 304–322 and 403–437 (Refs 26–28). In a recent study, Nagvekar et al. isolated Th0 clones specific for two distinct human AChR-a peptides, 75–90 and 149–158, in MG patients29. These observations indicate that the roles of Th1 (IFN-g-producing) and Th2 (IL-4-producing) subsets in anti-AChR Ab production and progression to disease are not as clear cut as was once thought.

A role for IFN-g rather than IL-4 in MG The involvement of the Th1 cytokine IFN-g in MG was explored by generating epsilon (e)-IFN-g-transgenic BALB/c mice30. Expressing the IFN-g (Ifng) gene in the neuromuscular junction under the control of the promoter from the gene encoding the murine nicotinic

AChR-e yielded unexpected results. These transgenic mice developed muscular weakness and flaccid paralysis as a result of functional disruption at the neuromuscular junction. The MG-like disease in these mice seemed to be B-cell dependent because the titer of autoAbs correlated with the severity of disease. Abs are deposited in the neuromuscular junctions of these mice. In these animals, the IgG response is not directed against mAChR, but against an undefined 87-kDa protein at the neuromuscular junction23. The mechanisms by which IFN-g favors pathogenic humoral immunity are unknown. Possibly, by virtue of its ability to induce costimulatory and MHC class II molecules, IFN-g might confer antigen-presenting capability on myocytes in the milieu of the neuromuscular junction, and thereby promote an antigen-specific Th1 response and autoAb response. Because autoAbs to mouse mAChRs were not elicited in these mice, this experimental model might represent the disease process occurring in seronegative MG patients. The requirement for IFN-g in the development of tAChR-induced disease has recently been examined in IFN-g-knockout (IFN-g-KO) mice31. After multiple immunizations with tAChR, IFN-g-KO mice fail to develop clinical EAMG. The inability of such mice to mount a crossreactive mAChR-specific response with IgG2a, IgG2b and IgG3 isotypes might account for their freedom from neuromuscular disease (Table 1). However, immunization of IFN-g-KO mice with keyhole limpet hemocyanin (KLH) resulted in decreased IgG2a titers, together with an increase in IgG1 titers. In other studies, a decrease in IFN-g production correlated with the suppression of EAMG after intraperitoneal administration of a146–162 peptide23 or nasal administration of a146–162, a181–200 and a360–378 peptides24 in mice that were challenged with tAChR. IFN-g is also an important participant in the rat model of EAMG, because the susceptibility or resistance of rats of disparate haplotypes to tAChR-induced EAMG correlates with the animals’ content of IFN-g-producing T cells32, and tAChR tolerization suppresses this disease in rats33,34. These findings show that IFN-g is required for the generation of a pathogenic anti-AChR humoral immune response and for the related clinical disease. Furthermore, the role of the Th1 cytokine IL-12 in EAMG has also been demonstrated35. In addition, tumor necrosis factor receptor 1 (TNFR1) signaling was found to be important in the

Table 1. Effect of cytokine/cytokine receptor gene disruption on tAChR-induced EAMG in mice T-cell responses to Mice IFN-g-knockout IL-12-knockout IL-4-knockout IFN-g-receptorknockout Wildtype

Mouse AChR-specific Abs

Clinical disease

tAChR

a146–162

AutoAbs

IgG1

IgG2a

IgG2b

IgG3

Refs

Absent Decreased Present Decreased

111 ND 111 1111

111 ND 111 ND

1/2 111 1

11 1111 1 111

1/2 1/2 111 1

1/2 ND 11 1

1/2 ND 11 1

31 35 36 53

Present

111

111

111

111

11

11

11

Abbreviations: AChR, acetylcholine receptor; autoAB, autoantibody; EAMG, experimental autoimmune myasthenia gravis; tAChR, Torpedo californica AChR; IFN-g, interferon g; IL-4, interleukin 4; ND, not done.

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(a) B7–CD28 costimulation pathway Initial priming

development of AChR-induced EAMG (H. KLH AChR Wang et al., pers. commun.). The contribution of the Th2 cytokine IL-4 to AChR-induced EAMG has also been exHigh-affinity Low-affinity amined. In contrast to the ability of IFN-g-KO T cell T cell mice immunized with tAChR to resist IFN-γ-dependent IFN-γ-dependent EAMG (Ref. 24), IL-4-KO mice readily develpathway pathway Dendritic cell oped this disease, with kinetics similar to IFN-γ that of the IL-4-sufficient counterparts. CD28 B7 These findings suggest that IL-4 is dispenB7 CD28 sable for the development of EAMG (Ref. 36), and that autoreactive T cells not producing Activated Resting Resting Activated IL-4 are capable of helping mAChR-specific Th cell B cell B cell Th cell B cells and of driving the pathogenic auto(high affinity) (low affinity) immune humoral response. IL-4-KO mice exhibited an increase in the levels of Th1 Humoral immune response cytokines and IgG2a titers (Table 1), but this enhancement in the Th1 cytokine profile was (b) CD27–CD27L costimulation pathway not followed by an acceleration of disease. IFN-γ + The IgG2b titers were similar between IL-4CD27 CD27L sufficient and IL-4-KO mice. Although IL-4 IFN-γ-dependent Humoral pathway seems nonessential in this situation, the immune response (AChR) findings do not rule out the possibility that Th1 cell Resting B cell IL-4 participates in the disease process of IL-4 wild-type mice, because immune sera from – tAChR-immunized mice contain IgG1 Abs CD27 CD27L IL-4-dependent (Th2 predominance) along with IgG2a and Impaired humoral pathway immune response IgG2b Abs (Th1 predominance). (AChR) Th2 cell Resting A recent study has suggested that IgG2b B cell Immunology Today Abs might exert myasthenogenic effects and thereby participate in the disease pathogenFig. 1. Hypothetical model for IFN-g-mediated immunomodulation of mouse AChR-specific autoesis because disease-susceptible C57BL/6 antibody response via (a) B7–CD28 and (b) CD27–CD27L costimulation pathways. Abbreviations: mice produce higher levels of mAChRAChR, acetylcholine receptor; CD27L, CD27 ligand; IFN-g, interferon g; IL-4, interleukin 4; KLH, specific IgG2b Abs than disease-resistant keyhole limpet hemocyanin; Th1, T helper 1. bm12 mutant mice20. The levels of IgG2a Abs remained very similar in these two strains of mice20. The fact that IFN-g, but not IL-4, exerts an etiopathogenic role IL-12 and the generation of Th1 cells because the production of the in Ab-dependent EAMG is somewhat counter-intuitive. Th1 cytokines IFN-g and IL-12 are interdependent39. In turn, this would dramatically affect the generation of complement-fixing IgG2a and IgG2b autoAbs. The interrelationship between disease Possible mechanisms: the role of IFN-g in modulation and IL-12 has been examined recently. Indeed, tAChR-immunized of the anti-AChR Ab response IL-12-KO mice are resistant to EAMG (Ref. 35). Finally, IFN-g might There are several possible mechanisms for the involvement of IFN-g operate via modulation of costimulation pathways (see below). in MG. First, IFN-g could upregulate MHC class II antigen on antiTo explain this latter possibility, this article proposes the hypogen-presenting cells, leading to effective presentation of T-cell epi- thetical model shown in Fig. 1. T cells responding to self-antigen(s) topes and the activation of T cells37. However, this scenario seems are generally of low affinity compared with those responding to forunlikely in the case of MG pathogenesis, because specific T-cell re- eign antigens. Expression of AChR in the thymus40 might enhance sponses are readily elicited in AChR-primed IFN-g-KO mice31. Sec- the negative selection of high-affinity AChR-reactive T cells, resultond, IFN-g could participate in EAMG by upregulating expression ing in a particularly low-affinity AChR T-cell repertoire. This is possof the complement factors C3 and C4 (Ref. 38), because IFN-g-KO ible because AChR expression is not strong enough in the thymus to mice are protected from the pathogenic features of complement- induce full tolerance, as addressed by experiments involving exfixing IgG2a and IgG2b autoAbs. This proposal also seems unlikely pression of a transgene encoding b-galactosidase in the murine because tAChR-primed IFN-g-KO mice barely mount an IgG2a and thymus41. During cognate T–B-cell interactions, these low-affinity IgG2b response. Third, the Ifng mutation might affect the release of AChR-specific T cells might require a relatively higher level of

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costimulation to achieve the threshold sufficient for contact-dependent signals to cause stimulation and differentiation of B cells and subsequent Ab production. According to this model, mounting an effective humoral immune response to mouse-AChR (self-antigen) would be dependent on IFN-g, which regulates expression of the costimulatory molecule B7-2 on B cells42. In support of this, a role for CD28–B7 signaling in the humoral immune response has been documented: treatment of mice with a human CTLA-4–immunoglobulin (Ig) fusion protein43 or an anti-B7-2 monoclonal antibody44 profoundly blocked the antibody response to a nominal antigen. This notion is further supported by the finding that transgenic expression of soluble murine CTL4-Hg1 in mice dramatically blocked humoral responses without affecting T-cell responses45. The importance of CD28–B7 interactions has been underscored by showing that EAMG is dramatically reduced in CD28-KO mice46. Because both Th1 and Th2 cytokines activate B cells to secrete antibodies, why would the Th1 pathway be essential for the development of MG? One possibility is through differential effects on the CD27–CD27L costimulatory pathway during cognate T–B-cell interactions47, as this pathway plays a crucial role in the humoral immune response. CD27 is a disulfide-linked 120-kDa transmembrane glycoprotein expressed on T and B cells that has homology to the tumor necrosis factor (TNF) family of molecules48. CD27–CD27L costimulation leads to B-cell activation and differentiation of B cells into Igproducing plasma cells49,50. An interesting recent study showed that the Th1 cytokine IFN-g upregulates, whereas IL-4 downregulates, the expression of CD27L on B cells, thereby differentially modulating the CD27–CD27L costimulation pathway51. Therefore, threshold levels of the Th1 cytokine IFN-g might promote CD27–CD27L costimulation during Th1–B-cell interactions, and favor the humoral immune response. In the absence of IFN-g, the relative increase in the IL-4 level might downregulate CD27L expression, leading to the protection of mice from tAChR-induced EAMG (Ref. 31). An impairment of CD27–CD27L costimulation during cognate T–B-cell interaction in AChR-immunized IFN-g-KO mice affects the synthesis of Th1-predominant IgG2a, IgG2b and IgG3 isotype production. Coincidentally, overexpression of the IL-4 (Il4) transgene by B cells completely prevented the development of lethal lupus-like glomerulonephritis by reducing the Th1-predominant IgG2a and IgG3 Abs in a murine model of systemic lupus erythematosus (SLE)52. The differential regulation of CD27L on B cells by Th1 (IFN-g) and Th2 (IL-4) cytokines might represent the point at which the Th1 and Th2 cytokine responses differentially modulate the pathogenic humoral immune response. Future studies should resolve this issue.

Concluding remarks The Th1/Th2 paradigm has provided a useful and simple model for defining the roles of Th-cell subsets in autoimmunity. Individual cytokines have exhibited paradoxical effects on the outcome of autoimmune diseases. Therefore, delineation of the role of individual cytokines is crucial for understanding particular immune responses in relation to autoimmune MG. The findings documented in mice

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with EAMG in relation to an essential role for IFN-g, but not for IL-4, might have important implications for designing immunotherapeutic strategies to treat MG patients.

We thank D. Kono, S. Gallichan, A. La Cava and F-D. Shi for their comments on this review. B.B. is supported by postdoctoral fellowships from the Juvenile Diabetes Foundation International (JDFI) and the Myasthenia Gravis Foundation of America. N.S. is supported by a diabetes interdisciplinary research program grant from the JDFI. This is manuscript no. 11791-IMM.

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