Keith A. Krolick

Immunological memory and late onset autoimmunity.

This review will address a paradox that has long fascinated scientists studying the effects of aging on the immune system. Although it has been clearly documented that B and T lymphocytes lose the ability to respond to antigenic or mitogenic stimulation with age, it has nonetheless been noted that the frequency of autoreactive antibodies is higher in older individuals. Given that the majority of the age-associated defects in immune regulation target the naïve T and B lymphocyte subsets, it has been presumed that this increase in antibodies specific for self antigens was due to changes in the B cell repertoire and/or to differences in the mechanisms responsible for generating immune tolerance in primary responses. However, in this review, we will address an alternative possibility that memory immune responses, first generated when the individual was young, may play a critical role in the appearance of serum autoantibodies by reactivation later in life (recall memory). It has recently been shown, in several different systems, that memory immunity can be maintained over the lifetime of the animal. Thus, memory B cells which are self-reactive may be harbored within an organism as it ages and the potential exists that they become re-activated at a later time, resulting in a vigorous autoreactive recall response. This may occur preferentially in older individuals due to several factors, including deficiencies in immune tolerance with age, progressive age-associated loss of tissue integrity yielding neo-self antigens, and possible re-exposure to an infectious agent which induces an autoimmune memory response through molecular mimicry. Thus, we propose that some of the autoantibodies seen in elderly patients and in older animals may have been produced by memory lymphocytes originally generated against antigens encountered during ones youth, but maintained in a tolerant (non reactive) state until a subsequent triggering event occurs. Possible implications of this model will be discussed.

Neoplastic B Cells as Targets for Antibody‐Ricin A Chain Immunotoxins

The targeting of toxic agents to tumor cells in vivo has been a goal of immunological research since the studies of Ehrlich (Himmelweit 1960). Successful application of this technique requires tumor-specific antibody whose activity remains unaltered following its covalent conjugation to a toxic agent. Moreover, the toxic portion of the conjugate should remain inactive until bound to the tumor cell via its antibody portion. In the studies described in this review, we have used an antibody against the cell surface immunoglobulin idiotype (Id) of a B cell tumor as our model system. B cell tumors are virtually always monoclonal in origin; hence each tumor cell that bears surface immunoglobulin expresses a particular Id (Fu et al. 1975. Schroer et al. 1974, Salsano et al. 1974). Since the Id is present on only a very small number of normal B cells (approximately l/lO*), the Id is operationally a tumor-specific antigen. Other advantages of using B cell tumors as model systems for antibody targeting studies include the availability of antibodies against other determinants on the cell surface immunoglobulin molecule (isotype, allotype, etc.) and a large body of information concerning the role of particular organs (e.g., spleen), cell types (T cells, macrophages) and lymphokines on the replication and differentiation of both normal and neoplastic B cells. In addition, a large number of humans with B cell tumors do not respond well to conventional chemo/radiotherapy (Lennert & Mohri 1978) and there is, therefore, a need to improve treatment. Recent studies utihzing antibody-directed targeting of toxic peptides to the surface of neoplastic cells are summarized in Table I and are reviewed in this volume. In general, the results of these studies have been promising. However,

Analysis of contractile properties of muscles from rats immunized with purified acetylcholine receptor.

Experimental autoimmune myasthenia gravis (EAMG) was induced in rats by injection of purified acetylcholine receptor (AChR). In addition to detecting elevated serum titers of anti-AChR antibodies, we observed decreased twitch-tension at submaximal stimulation voltages and increased curare sensitivity by muscles obtained from immunized rats when compared to muscles obtained from nonimmune control rats. Furthermore, antibody-induced neuromuscular impairment was expressed to differing extents dependent on whether the diaphragm, soleus, or extensor digitorum longus muscle was examined. Thus, we conclude that potential antibody perturbation of AChR function will depend not only on the nature of the antibody, but also on the complex structure-function relationships that exist in individual muscles. This may partially explain the variable impairment of different muscle groups in patients with myasthenia.

Acetylcholine receptor-reactive antibodies in experimental autoimmune myasthenia gravis differing in disease-causing potential: Subsetting of serum antibodies by preparative isoelectric focusing

Antibodies obtained from the sera of Lewis rats demonstrating impaired neuromuscular function following immunization with purified acetylcholine receptor (AChR) were fractionated by preparative isoelectric focusing. Upon passive transfer of fractionated anti-receptor antibodies into immunologically naive, healthy recipient rats it was observed that two main subsets of AChR-specific antibody could be identified. One subset, representing about one-third of the expressed clonotypic antibody repertoire, was capable of directly perturbing AChR-dependent neuromuscular function following transfer. A second subset, demonstrated no detectable ability to induce disease symptoms following transfer. Although the anti-AChR antibodies were produced by immunization with Torpedo AChR, the inability of some antibody fractions to perturb AChR function was not explained by their inability to react with AChR of mammalian origin. Furthermore, the ability to transfer symptoms did not correspond with a particular antibody isotype (although the response was dominated by IgG2a) and did not depend solely on high relative binding avidity (benign reactivities of high relative binding avidity were also observed). Nonetheless, an anti-AChR antibody subset can be directly identified and purified from immune serum that is likely to contain reactivities that are most directly responsible for neuromuscular disease symptoms demonstrated by rats with experimental autoimmune myasthenia gravis.

Influence of T cell specificity on the heterogeneity and disease-causing capability of antibody against the acetylcholine receptor

Abstract Adoptive secondary anti-acetylcholine receptor (AChR) antibody responses were examined in rats to evaluate the influence of helper T cell specificity on the nature and disease-causing potential of antibody produced. Mixtures of B cells reactive with the intact AChR plus T cells reactive with purified AChR subunits (α, β, γ, δ) were transferred and antigen-challenged in immunologically naive recipient rats; the serum anti-AChR antibody produced was assessed by radioimmunoassay for differences in titers and by isoelectric focusing for differences in clonal heterogeneity as a function of the subunit specificity of T cells transferred. In addition, rats receiving different sources of AChR or AChR subunit-reactive T cells were examined for AChR-dependent muscle dysfunction. The results indicated a clear reduction in anti-AChR antibody concentrations and clonal heterogeneity in recipient rats receiving T cells of specificities restricted to individual subunits. However, except for a clear relationship between serum anti-AChR antibody concentration and disease induction, no particular AChR subunit-reactive helper T cell specificity appeared to preferentially cause muscle dysfunction. We conclude that if such relationships exists, T cells with specificities more restricted than those described here will have to be used.

Muscle Responds to an Antibody Reactive with the Acetylcholine Receptor by Up-Regulating Monocyte Chemoattractant Protein 1: A Chemokine with the Potential to Influence the Severity and Course of Experimental Myasthenia Gravis

Autoantibodies with reactivity against the postjunctional muscle receptor for acetylcholine receptor are able to interfere with contractile function of skeletal muscles and cause the symptoms of myasthenia gravis (MG) in humans, as well as in experimental animal models of MG. In the study described below using a rat model of MG, it was observed that exposure to acetylcholine receptor-reactive Abs also induced increased levels of chemokine (i.e., monocyte chemoattractant protein 1) production by skeletal muscle cells. This was true of both cultured rat myocytes exposed in vitro and rat muscle exposed in vivo following passive Ab transfer. Increased monocyte chemoattractant protein 1 production may explain the increased trafficking of leukocytes through muscle following Ab transfer described in this and other reports. These observations may also be relevant to the induction of disease symptoms in experimental animal models of MG, since numerous reports from this and other laboratories indicate that the cytokine environment provided by leukocytes trafficking through muscle may play a pivotal role in disease progression.

Examination of characteristics that may distinguish disease-causing from benign AChR-reactive antibodies in experimental autoimmune myasthenia gravis

In summary, the strategies of the experimentation described above were designed to address the confusion resulting from observations concerning the lack of correlation between antibody titers and disease severity in MG patients. Lessons learned from these studies of EAMG suggest that if the proportion of the total expressed/produced anti-AChR antibody repertoire with disease-causing potential differs from patient-to-patient with MG, then assessment of the total antibody titer becomes meaningless unless a particular patient produces disease-causing reactivities that make up a major portion of the total titer. Not only may disease severity depend on the titer of a small subset of disease-causing antibody(s) reactive with a particular conformation-dependent AChR region, but may also depend on the relative contribution of additional subsets of antibody with functionally irrelevant or potentially protective activity. The key to exploiting the existence of antibody subsets with differing disease-causing potential will be to create probes that would allow the easy monitoring of the relevant reactivities. For instance, carefully selected anti-idiotypic antibodies (such as the 11E10 monoclonal antibody described above) may be of great value when specifically capable of recognizing idiotypes that are selectively associated with disease-causing anti-AChR antibodies and under-represented on antibodies lacking disease-causing capability. If, in addition, characteristics of helper T cells are identified that allow more accurate prediction of D+ Id production, exciting opportunities would become available to more directly evaluate disease mechanisms and to develop more highly efficacious immunotherapeutic strategies.

Clonotypic analysis of the antibody response to the acetylcholine receptor in experimental autoimmune myasthenia gravis

Autoreactive B cells reactive with the acetylcholine receptor (AChR), and the antibodies produced by them, are proposed to play a primary role in the immunopathology of myasthenia gravis and its animal models. Therefore, the anti-AChR antibody response induced in rats was characterized for the clonotypic heterogeneity, isotype distribution, and affinity by isoelectric focusing (IEF) and affinity immunoblotting. It was determined that the rat anti-AChR serum antibody was relatively heterogeneous, reflecting the oligoclonality of the response. Furthermore, isotypic dominance by IgG2a was observed in that the majority of clonal products detected by IEF were of this isotype in both primary and secondary responses. Lastly, the clonotypic anti-AChR antibodies were of relatively low affinity (avidity) when compared to antibodies reactive with the highly immunogenic protein antigen, keyhole limpet hemocyanin; anti-AChR antibody avidity did not appear to increase when the antibodies in the secondary response were compared to antibodies in the primary response. These antibody characteristics are discussed in terms of their role in disease induction.

Split Tolerance in a Novel Transgenic Model of Autoimmune Myasthenia Gravis

Because it is one of the few autoimmune disorders in which the target autoantigen has been definitively identified, myasthenia gravis (MG) provides a unique opportunity for testing basic concepts of immune tolerance. In most MG patients, Abs against the acetylcholine receptors (AChR) at the neuromuscular junction can be readily identified and have been directly shown to cause muscle weakness. T cells have also been implicated and appear to play a role in regulating the pathogenic B cells. A murine MG model, generated by immunizing mice with heterologous AChR from the electric fish Torpedo californica, has been used extensively. In these animals, Abs cross-react with murine AChR; however, the T cells do not. Thus, to study tolerance to AChR, a transgenic mouse model was generated in which the immunodominant Torpedo AChR (T-AChR) α subunit is expressed in appropriate tissues. Upon immunization, these mice showed greatly reduced T cell responses to T-AChR and the immunodominant α-chain peptide. Limiting dilution assays suggest the likely mechanism of tolerance is deletion or anergy. Despite this tolerance, immunization with intact T-AChR induced anti-AChR Abs, including Abs against the α subunit, and the incidence of MG-like symptoms was similar to that of wild-type animals. Furthermore, evidence suggests that this B cell response to the α-chain receives help from T cells directed against the other AChR polypeptides (β, γ, or δ). This model offers a novel opportunity to elucidate mechanisms of tolerance regulation to muscle AChR and to clarify the role of T cells in MG.

Antigen presentation and T cell specificity repertoire in determining responsiveness to an epitope important in experimental autoimmune myasthenia gravis

The overall goal of this study was to define, in experimental autoimmune myasthenia gravis (EAMG), immunological differences between helper T cells from two inbred rat strains that explain disease susceptibility on the one hand (in Lewis rats) and disease resistance on the other hand (in Wistar Furth rats). The working hypothesis for these studies was that the T cell compartment in Wistar Furth rats may lack the ability to activate responsiveness by existing B cells that have the potential to produce disease-causing antibodies; this quality may be related to a lack of T cell reactivity toward a determinant(s) associated with the alpha subunit sequence alpha 100-116 of the acetylcholine receptor (AChR) that demonstrates immunodominance in Lewis rats. Results presented below are consistent with the conclusion that there is a deficit in the Wistar Furth (WF) T cell specificity repertoire responsible for unresponsiveness to this important AChR epitope; this deficit exists despite the apparent ability of WF antigen presenting cells to bind and present the alpha 100-116 peptide, and is likely responsible for the inability of Wistar Furth T cells to drive an anti-AChR antibody response with disease-causing potential.