T cell recognition of the A2 domain of coagulation factor VIII in hemophilia patients and healthy subjects.

Hemophilia A patients treated with coagulation factor VIII (FVIII), and also some healthy subjects, may develop anti-FVIII antibodies (Ab), whose synthesis is driven by FVIII-specific CD4+ T cells. Some Ab block the procoagulant function of FVIII (inhibitors). Many inhibitors recognize epitopes on the FVIII A2 domain. Here, we have sought to identify A2 epitopes recognized by CD4+ T cells. We tested the proliferative response of CD4+ blood lymphocytes (BL) from hemophilia patients and healthy subjects, to overlapping synthetic peptides spanning the A2 domain sequence. Many A2 peptides induced proliferative responses of CD4+ BL from one or more subjects. The peptide-induced responses were strongest in hemophilia patients with inhibitors, weakest in healthy subjects. A2 peptides comprising residues 371-400, 621-650 and 671-690 elicited frequent and strong responses in hemophilia A patients, and especially in those with inhibitors. Healthy subjects recognized frequently only the sequence 371-400. A three-dimensional model of the A2 domain suggests that these CD4+ epitope sequences have structural features typical of 'universal' CD4+ T epitopes.

Epitope repertoire of human CD4(+) T cells on the A3 domain of coagulation factor VIII.

Severe hemophilia A patients treated with factor (F)VIII may develop antibodies (Ab) that block FVIII function (inhibitors). Autoimmune inhibitors may develop in subjects without congenital hemophilia, and cause acquired hemophilia. Hemophiliacs without inhibitors and healthy subjects may also have small amounts of antiFVIII Ab. FVIII-specific CD4(+) T cells induce antiFVIII Ab synthesis. Here, we have examined their epitope repertoire in hemophilia patients and healthy subjects. We used overlapping synthetic peptides, spanning the sequence of the FVIII A3 domain, to challenge blood CD4(+) T cells in proliferation assays. The epitopes recognized in hemophilia A patients with or without inhibitors, acquired hemophilia patients, or healthy subjects overlapped, yet had characteristic differences. Most members of one or more study groups recognized the sequence regions 1691-1710, 1801-1820, 1831-1850, and 1941-60. In the proposed three-dimensional structure of the A3 domain, these sequences are largely exposed to the solvent and flanked by flexible sequence loops: these are structural features characteristic of 'universal' CD4(+) T epitopes. Hemophilia A patients with inhibitors recognized prominently only the sequence 1801-1820, which overlaps a known inhibitor binding site. This is consistent with the possibility that CD4(+) T cells recognizing epitopes within residues 1801-1820 have a role in inducing inhibitor synthesis. In contrast, CD4(+) T cells sensitized to sequences 1691-1710 and 1941-60, which are recognized by healthy subjects and hemophilia A patients without inhibitors, might curb inhibitor synthesis.

Characterization of human hybridoma clones isolated from hemophilia patients with specificity for different domains of coagulating factor VIII.

Hemophilia A patients treated with human coagulating factor VIII (FVIII) may develop inhibitory antibodies (inhibitors). Characterization of the inhibitors at the clonal level may help exploring new therapeutic strategies. We have generated lymphoblastoid cell lines (LCLs) producing anti-FVIII antibodies from peripheral blood lymphocytes of hemophilia A patients with high inhibitor titers. We fused the anti-FVIII-positive LCLs with a heteromyeloma, to produce FVIII specific hybridomas. We determined the specificity, isotype, idiotypic and immunoglobulin (Ig) variable region heavy (VH) chain gene family profiles of the secreted antibodies (Ab) by ELISA, immunoblotting and RT-PCR. We established eight hybridomas which produced high titers of anti-FVIII Ab. All hybridomas secreted IgM Ab, associated with either kappa(5/8) or lambda(3/8) light chain. Analysis of the expressed VH genes by RT-PCR revealed that the hybridomas utilized only the VH1 (63%) or the VH3 (37%) gene families. Among the cross-reactive idiotypes (CRIs) we tested, only the VH1 and VK3b-associated CRIs were expressed by 3 hybridomas. Immunoblotting of thrombin-digested FVIII demonstrated distinct patterns of reactivity of the monoclonal Ab (MAb) secreted by the hybridomas, which recognized either the A2 domain of the Fvm heavy chain, or the light chain, or both. Our findings suggest that: a) the isotype of the anti-FVIII Ab secreted by LCLs and hybridoma clones (IgM) differs from that of anti-FVIII Ab in vivo, which are predominantly IgG4: this suggests a negative selection of the isotype-switched FVIII-specific B-cells in the periphery of these patients; b) the anti-FVIII Ab have a biased representation of the VH1 gene family, and c) somatic mutations in the VH genes coding for FVIII specificity occur in the anti-FVIII Ab response, as evidenced by lack of expression of the VH-associated CRI.

Recognition of coagulation factor VIII by CD4+ T cells of healthy humans.

Hemophilia A patients treated with coagulation factor (F)VIII may develop an anti-FVIII immune response. Anti-FVIII antibodies may occur also in healthy subjects. To understand the extent to which an immune response to FVIII occurs in healthy subjects, we investigated the proliferative response of blood CD4+ T cells from 90 blood donors to FVIII and to pools of overlapping synthetic peptides spanning the sequences of individual FVIII domains (A1-A3, C1-C2). Most subjects responded to FVIII and several FVIII domains. Men had stronger responses to FVIII than women, and older subjects than younger subjects. The domain-induced responses were weaker than the FVIII-induced responses, yet their intensity in individual subjects correlated with that of the response to FVIII. We examined whether Th1 and/or Th2 cells responded to FVIII in 68 subjects, by determining the CD4+ T cells that secreted interferon-gamma (IFN-gamma) or interleukin (IL)-5 after stimulation with FVIII: 25 subjects had FVIII-specific IFN-gamma-secreting cells, and seven of them had also FVIII-specific IL-5-secreting cells. None had only IL-5-secreting cells. Thus, a CD4+ T cell response to FVIII, which first involves Th1 cells, is common among subjects with a normal procoagulant function.

Human CD4+ T-cell epitope repertoire on the C2 domain of coagulation factor VIII.

Approximately 25% of severe hemophilia A patients develop antibodies (Ab) that neutralize the procoagulant function of factor (F)VIII (inhibitors). Autoimmune FVIII inhibitors may develop in individuals without congenital FVIII deficiency and cause acquired hemophilia. Low titers of anti-FVIII Ab may be present in hemophilia A patients without inhibitors and in healthy blood donors. FVIII-specific CD4+ T-cells drive the synthesis of anti-FVIII Ab. We examined the epitope repertoire of CD4+ T-cells from 15 healthy subjects, 10 hemophilia A patients without inhibitors, 11 hemophilia A patients with inhibitors, and six acquired hemophilia patients. Blood CD4+ T-cells were challenged in proliferation assays with a panel 16 overlapping synthetic peptides, spanning the sequence of the FVIII C2 domain. The sequence region 2291-2330 contained the most frequently and strongly recognized peptides in each of the four subject groups. Crystallographic B factor data and the location of these peptides within the three-dimensional structure of the C2 domain confirm that this region has a high degree of solvent exposure and flexibility within the peptide backbone, which are structural features typical of immunodominant universal CD4+ epitopes. Furthermore, this sequence region overlaps inhibitor-binding sites, suggesting that CD4+ T-cells recognizing peptide sequences within this region might be involved in inhibitor synthesis. The sequence regions 2191-2210 (recognized strongly by each study group except hemophilia A patients with inhibitors) and 2241-2290 (recognized primarily by acquired hemophilia patients and healthy subjects) share the same structural features, and also overlap inhibitor-binding sites. Although similar, there appear to be important differences in the CD4+ epitope repertoires of congenital and acquired hemophilia patients.

Human bronchial epithelial and endothelial cells express alpha7 nicotinic acetylcholine receptors.

The epithelial or endothelial cells that line the human bronchi and the aorta express nicotinic acetylcholine receptors (nAChRs) of alpha3 subtypes. We report here that human bronchial epithelial cells (BEC) and aortic endothelial cells (AEC) express also the nAChR alpha7 subunit, which forms functional nAChRs. Polymerase chain reaction and in situ hybridization experiments detected alpha7 subunit mRNA in cultured human BEC and AEC and in sections of rat trachea. The binding of radiolabeled alpha-bungarotoxin revealed a few thousand binding sites per cell in cultured human BEC and human and bovine AEC. Western blot and immunohistochemistry experiments demonstrated that cultured BEC and AEC express a protein(s) recognized by anti-alpha7 antibodies. Whole-cell patch-clamp studies of cultured human BEC demonstrated the presence of fast-desensitizing currents activated by choline and nicotine that were blocked reversibly by methyllycaconitine (1 nM) and irreversibly by alpha-bungarotoxin (100 nM), consistent with the expression of functional alpha7 nAChRs. In some cells, choline activated also slowly decaying currents, confirming previous reports that BEC express functional alpha3beta4 nAChRs. Exposure of cultured BEC to nicotine (1 microM) for 3 days up-regulated functional alpha7 and alpha3 nAChRs, as indicated by the increased number of cells responding to acetylcholine and choline, with both fast-desensitizing currents, which were blocked irreversibly by alpha-bungarotoxin, and with slowly desensitizing currents, which are alpha-bungarotoxin-insensitive currents. The presence of alpha7 nAChRs in BEC and AEC suggests that some toxic effects of tobacco smoke could be mediated through these nicotine-sensitive receptors.

CD4+ T cells specific for factor VIII as a target for specific suppression of inhibitor production.

The studies we reviewed here have begun to clarify the complex cellular mechanisms involved in the immune response to fVIII, and the circumstances under which fVIII inhibitors develop. Further characterization and comparison of the immune response to fVIII in both hemophilia patients and healthy subjects will help to further elucidate these mechanisms. The murine hemophilia model will hopefully provide further insights into the mechanisms of inhibitor formation, and prove to be a suitable tool for the design and testing of therapeutic strategies aimed at preventing the development of fVIII inhibitors.

Transgenic expression of IL-10 in T cells facilitates development of experimental myasthenia gravis.

Ab to the acetylcholine receptor (AChR) cause experimental myasthenia gravis (EMG). Th1 cytokines facilitate EMG, whereas Th2 cytokines might be protective. IL-10 inhibits Th1 responses but facilitates B cell proliferation and Ig production. We examined the role of IL-10 in EMG by using wild-type (WT) C57BL/6 mice and transgenic (TG) C57BL/6 mice that express IL-10 under control of the IL-2 promoter. We immunized the mice with doses of AChR that cause EMG in WT mice or with low doses ineffective at causing EMG in WT mice. After low-dose AChR immunization, WT mice did not develop EMG and had very little anti-AChR serum Ab, which were mainly IgG1, whereas TG mice developed EMG and had higher levels of anti-AChR serum Ab, which were mainly IgG2, in addition to IgG1. At the higher doses, TG mice developed EMG earlier and more frequently than WT mice and had more serum anti-AChR Ab. Both strains had similar relative serum concentrations of anti-AChR IgG subclasses and IgG and complement at the muscle synapses. CD8(+)-depleted splenocytes from all AChR-immunized mice proliferated in the presence of AChR and recognized a similar epitope repertoire. CD8(+)-depleted splenocytes from AChR-immunized TG mice stimulated in vitro with AChR secreted significantly more IL-10, but less of the prototypic Th1 cytokine IFN-gamma, than those from WT mice. They secreted comparable amounts of IL-4 and slightly but not significantly reduced amounts of IL-2. This suggests that TG mice had reduced activation of anti-Torpedo AChR Th1 cells, but increased anti-AChR Ab synthesis, that likely resulted from IL-10-mediated stimulation of anti-AChR B cells. Thus, EMG development is not strictly dependent on Th1 cell activity.

Human thymuses express incomplete sets of muscle acetylcholine receptor subunit transcripts that seldom include the delta subunit.

In myasthenia gravis (MG) the muscle acetylcholine receptor (AChR) is the target of an immune response that might begin in the thymus. The thymus expresses binding sites for specific ligands of muscle AChR, a complex protein composed of alpha, beta, gamma (or epsilon) and delta subunits. The thymus expresses the AChR alpha subunit, but there is controversy regarding the expression in the thymus of the gamma, epsilon and delta subunits. We investigated the presence of messenger RNA (mRNA) for the different muscle AChR subunits in thymus tissue from 20 healthy subjects and 13 myasthenic patients. We detected mRNA for the alpha and epsilon subunits in all samples, for the beta subunit in all but one sample and for the gamma subunit in most samples although at lower levels than the epsilon subunit. Myasthenic thymuses expressed levels of gamma subunit mRNA similar to control thymuses but more abundant epsilon subunit mRNA. None of the myasthenic thymuses and only two control thymuses expressed detectable delta subunit mRNA. This supports the hypothesis that human thymus may express AChR proteins that do not include the delta subunit. Such receptors, which would have different antigenic structure than the muscle AChRs, might have a role in triggering the autoimmune response that causes MG.

Mechanism of the immune response to human factor VIII in murine hemophilia A.

Mice genetically deficient in factor VII (fVIII) are a model of hemophilia A. As a first step to reproduce in this mouse model what occurs over time in hemophilia A patients treated with human fVIII (hfVIII), we have investigated the time course and the characteristics of their immune response to hfVIII, after multiple intravenous injections. Anti-hfVIII antibodies appeared after four to five injections. They were IgG1 and to a lesser extent IgG2, indicating that they were induced by both Th2 and Th1 cells. Inhibitors appeared after six injections. CD4+ enriched splenocytes from hfVIII-treated mice proliferated in response to fVIII and secreted IL-10: in a few mice they secreted also IFN-gamma and in one mouse IL-4, but never IL-2. A hfVIII-specific T cell line derived from hfVIII-treated mice secreted both IL-4 and IFN-gamma, suggesting that it included both Th1 and Th2 cells. CD4+ enriched splenocytes of hfIII-treated mice recognized all hfVIII domains. Thus, hemophilic mice develop an immune response to hfVIII administered intravenously similar to that of hemophilia A patients. Their anti-hfVIII antibodies can be inhibitors and belong to IgG subclasses homologous to those of inhibitors in hemophilic patients; their anti-hfVIII CD4+ cells recognize a complex repertoire and both Th1 and Th2 cytokines, and especially IL-10, may drive the antibody synthesis.

Sensitization of CD4+ T cells to coagulation factor VIII: response in congenital and acquired hemophilia patients and in healthy subjects.

Antibodies (Ab) that inhibit factor VIII (fVIII) may develop in patients with hemophilia A and rarely in individuals without congenital fVIII deficiency (acquired hemophilia). Synthesis of fVIII inhibitors requires CD4+ T cells. We investigated the proliferative response of blood CD4+ cells from 11 patients with congenital or acquired hemophilia and 12 healthy subjects, to recombinant human fVIII, and to pools of overlapping synthetic peptides spanning the sequences of individual fVIII domains. All patients had CD4+ cells that responded to fVIII. The intensity of the responses fluctuated over time: several patients had brief periods when they did not respond to fVIII. All healthy subjects had transient CD4+ responses to fVIII, that were significantly lower than those of hemophilia patients. Also, healthy subjects responded to fVIII less frequently and for shorter periods than hemophilia patients. All patients and healthy subjects recognized several fVIII domains: the A3 domain was recognized most strongly and frequently. The transient sensitization of CD4+ cells to fVIII in healthy subjects suggests that inadequate tolerization of CD4+ cells to fVIII, due to lack of endogenous fVIII, is an important factor in the development of clinically significant anti-fVIII antibodies in hemophilia A.

T cell recognition of muscle acetylcholine receptor in ocular myasthenia gravis.

We examined the proliferative response of blood CD4(+) cells to muscle acetylcholine receptor (AChR) subunits and the epitope repertoire of the epsilon and gamma subunits, in ocular myasthenia gravis (oMG) patients and healthy subjects. oMG patients seldom recognized all subunits. The frequency and intensity of recognition was the same for all subunits, irrespective of the disease duration. The responses in oMG were lower than in generalized myasthenia gravis. Healthy subjects had frequent, low responses to one or more subunits. oMG patients recognized several epitopes on the gamma and epsilon subunits, that partially overlapped those recognized in gMG. The subunits and epitopes recognized by individual oMG patients changed over time. Thus, oMG patients have minimal and unstable sensitization of anti-AChR CD4(+) cells, in agreement with their low and inconsistent synthesis of anti-AChR antibody.

Absence of IFN-gamma or IL-12 has different effects on experimental myasthenia gravis in C57BL/6 mice.

Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). Th1 cells facilitate EMG development. IFN-gamma and IL-12 induce Th1 responses: we investigated whether these cytokines are necessary for EMG development. We immunized wild-type (WT) C57BL/6 mice and IFN-gamma and IL-12 knockout mutants (IFN-gamma-/-, IL-12-/-) with Torpedo AChR (TAChR). WT and IFN-gamma-/- mice developed EMG with similar frequency, IL-12-/-mice were resistant to EMG. All strains synthesized anti-AChR Ab that were not IgM or IgE. WT mice had anti-AChR IgG1, IgG2b, and IgG2c, IFN-gamma-/- mice had significantly less IgG2c, and IL-12-/- mice less IgG2b and IgG2c. All mice had IgG bound to muscle synapses, but only WT and IFN-gamma-/- mice had complement; WT mice had both IgG2b and IgG2c, IFN-gamma-/- only IgG2b, and IL-12-/- neither IgG2b nor IgG2c. CD4+ cells from all AChR-immunized mice proliferated in response to AChR and recognized similar epitopes. After stimulation with TAChR, CD4+ cells from IFN-gamma-/- mice secreted less IL-2 and similar amounts of IL-4 and IL-10 as WT mice. CD4+ cells from IL-12-/- mice secreted less IFN-gamma, but more IL-4 and IL-10 than WT mice, suggesting that they developed a stronger Th2 response to TAChR. The EMG resistance of IL-12-/- mice is likely due to both reduction of anti-TAChR Ab that bind complement and sensitization of modulatory Th2 cells. The reduced Th1 function of IFN-gamma-/- mice does not suffice to reduce all complement-fixing IgG subclasses, perhaps because as in WT mice a protective Th2 response is missing.

Neuronal nicotinic receptors in non-neuronal cells: new mediators of tobacco toxicity?

The nicotinic acetylcholine receptors are prototypic ionotropic receptors that mediate fast synaptic transmission. However, also non-excitable cells, and particularly the tegumental cells that line external and internal body surfaces, express acetylcholine receptors of neuronal type sensitive to nicotine. Bronchial epithelial cells, endothelial cells of blood vessels and skin keratinocytes express neuronal nicotinic receptors composed of alpha(3), alpha(5), beta(2) and beta(4) subunits, similar to those expressed in sympathetic ganglia, and neuronal nicotinic receptors composed of alpha(7) subunits. Neuronal nicotinic receptors in tegumental cells are involved in modulating cell shape and motility, and therefore in maintaining the integrity of the surfaces lined by those cells. Neuronal nicotinic receptors in non-neuronal tissues may modulate other functions, including cell proliferation and differentiation. Acetylcholine is synthesized, secreted and degraded by a variety of cells, including the tegumental cells that express neuronal nicotinic receptors. Thus, acetylcholine may function as a local "hormone" that is able to modulate cell functions that require fast adaptation to new conditions. The presence of neuronal nicotinic receptors sensitive to nicotine in tissues known to be involved in tobacco toxicity, like bronchi and blood vessels, raises the possibility that they mediate some of the toxic effects of smoking.

Universal epitopes for human CD4+ cells on tetanus and diphtheria toxins.

Previous studies suggested that tetanus and diphtheria toxoids (TTD and DTD, respectively) contain "universal" epitopes for human CD4+ cells (residues 632-651 and 950-969 of TTD and 271-290, 321-350, 351-370, 411-430, and 431-450 of DTD). To investigate whether CD4+ cells of 100 randomly selected subjects recognized those sequences, the proliferation of CD4+ cell-enriched blood lymphocytes to TTD and DTD and individual synthetic universal epitopes was measured. CD4+ cells of 98 subjects recognized both toxoids, those of 1 subject only TTD, and those of 1 only DTD. The TTD peptides and DTD peptides 271-290 and 331-350 were recognized by >/=80% of the toxoid-sensitized subjects. The other DTD sequences were recognized by 63%-71% of subjects. DR-homozygous subjects recognized several universal epitopes less frequently than did DR-heterozygous subjects. The intensity of responses to the epitope peptides correlated with that to TTD or DTD, consistent with recognition of the peptides by CD4+ cells specific for the cognate toxoid.

CD4+ T cell response to factor VIII in hemophilia A, acquired hemophilia, and healthy subjects.

These studies have begun to clarify the complex cellular mechanisms involved in the immune response to factor VIII. Although vigorous sensitization of CD4+ cells occurs in healthy subjects, the absence of clinically significant levels of inhibitor antibodies is likely related to the prompt down-regulation of the immune response. It may also be possible that the specific epitope repertoire recognized by CD4+ cells plays a role in the outcome of the immune response to factor VIII. Further characterization and comparison of the CD4+ repertoire in healthy subjects with that of hemophilia patients with and without inhibitors will help clarify which mechanism explains the absence of productive inhibitor synthesis in certain individuals. Also, it might identify CD4+ epitopes recognized by T helper cells that are essential for inhibitor synthesis. Additional studies to further characterize the role of Th1 and Th2 cells in the immune response to factor VIII may also be needed for the design of novel therapeutic strategies aimed at preventing inhibitor development.

Subcutaneous administration of T-epitope sequences of the acetylcholine receptor prevents experimental myasthenia gravis.

Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). The s.c. administration to C57B1/6 mice of synthetic AChR CD4+ epitopes, before and during AChR immunization, reduced the epitope-specific CD4+ responses and the anti-AChR Ab synthesis, and prevented EMG. The s.c. administration of solubilized AChR had effects similar to those of peptide treatment. Sham-tolerized mice had only Th1 anti-AChR cells, whereas peptide-treated mice had also Th2 cells, and Th2-induced anti-peptide Ab. Established EMG was not affected by s.c. peptide treatment, whereas it worsened after s.c. administration of solubilized AChR.

Interleukin-4 deficiency facilitates development of experimental myasthenia gravis and precludes its prevention by nasal administration of CD4+ epitope sequences of the acetylcholine receptor.

Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). We investigated EMG in interleukin (IL)-4 knock out B6 (KO) mice, that lack Th2 cells. EMG was more frequent in KO than in wild type B6 mice. KO and B6 mice developed similar amounts of anti-AChR antibodies. They were IgG2a and IgG2b in KO mice, IgG1 and IgG2b in B6 mice. CD4+ cells from KO and B6 mice recognized the same AChR epitopes. Nasal administration of synthetic AChR CD4+ epitopes reduced antibody synthesis and prevented EMG in B6, not in KO mice. Thus, Th2 cells may have protective functions in EMG.

Myasthenia in SCID mice grafted with myasthenic patient lymphocytes: role of CD4+ and CD8+ cells.

OBJECTIVES: Acetylcholine receptor (AChR)-specific CD4+ cells are present in MG patients, and synthesis of the high-affinity immunoglobulin G (IgG) autoantibodies (autoAb) against the muscle AChR that causes MG symptoms requires intervention of CD4+ cells. The role of CD4+ cells in MG pathogenesis has been postulated but never proven. MG patients do not have anti-AChR cytotoxic phenomena, and it has been assumed that CD8+ cells do not have a pathogenic role in MG. However, CD8+ cells may facilitate rodent experimental MG, raising the possibility that CD8+ cells might be necessary also in MG. In this study we examined whether CD4+ and CD8+ cells play a role in the pathogenesis of MG and whether CD4+ cells specific for AChR epitope sequences recognized by most MG patients ("universal" epitopes) drive the synthesis of pathogenic antibodies. METHODS: First we characterized a chimeric human-mouse model of MG in severe combined immunodeficiency (SCID) mice engrafted with blood lymphocytes (BL) from MG patients. We used that model to determine whether CD4+ and CD8+ cells are necessary for transfer of MG symptoms. We engrafted SCID mice intraperitoneum with BL from 19 MG patients and 5 healthy controls. We engrafted some mice with either BL, BL depleted in CD4+ or CD8+ cells from the same patient, or CD4+ depleted BL reconstituted with CD4+ T cells from the same patient, specific for "universal" AChR epitopes or for two unrelated antigens, tetanus and diphtheria toxoids. We tested the mice for myasthenic symptoms for 7 to 18 weeks. RESULTS: Mice transplanted with BL, or CD8+ depleted BL, or CD4+-depleted BL reconstituted with anti-AChR CD4+ cells from MG patients frequently developed myasthenic weakness. The mice had human anti-AChR Ab in the serum and bound to muscle AChR. Mice transplanted with BL from controls, or CD4+-depleted BL from MG patients, or CD4+-depleted BL from an MG patient reconstituted with CD4+ cells specific for tetanus or diphtheria toxoids did not develop myasthenic weakness or anti-AChR Ab. CONCLUSIONS: CD4+ cells are necessary for MG pathogenesis; CD8+ cells may not be. CD4+ cells specific for "universal" AChR epitopes help the synthesis of pathogenic Ab.