S. Cohen-Kaminsky

Identification of genomic typing of non-DR3 HLA class II genes associated with myasthenia gravis

HLA association with myasthenia gravis (MG) has been studied in a series of 114 patients using class I and class II genotyping after PCR amplification. Positive association was found with DR3, particularly in women (RR = 2.6) and in early MG onset (RR = 3.4). DRB1, DRB3, DQB1, DQA1 and B (B8 and B18) genotyping revealed that the association was predominantly with the B8 DRB1*03 DRB3*0101 DQB1*0201 DQA1*0501 ancestral haplotype. This haplotype frequency was also increased in patients with thymic hyperplasia (RR = 3.5) and was greatly reduced in patients with thymoma (RR = 0.35). Sixteen out of 48 patients carrying this 8.1 ancestral haplotype showed absence of B8 (n = 4) or of DR3 (n = 12). HLA class II genotyping further revealed the existence of two other significant associations. MG was positively associated with the DQB1*0604 allele (RR = 3.4), particularly in patients with thymoma (RR = 5.7). Furthermore, the disease was negatively associated with DR1 in females (RR = 0.32). These data suggest that MG is placed under the control of at least three distinct genes: (1) a class II predisposing gene in the 8.1 ancestral haplotype; (2) a thymoma-associated class II allele on the DQB1*0604 haplotype; and (3) a protective allele DR1.

Prevention of autoimmune attack by targeting specific T-cell receptors in a severe combined immunodeficiency mouse model of myasthenia gravis

Myasthenia gravis (MG) is an autoimmune disease targeting the skeletal muscle acetylcholine receptor. We have previously demonstrated a selection bias of CD4+ T cells expressing the Vβ5.1 T‐cell receptor gene in the thymus of HLA‐DR3 patients with MG. To evaluate the pathogenicity of these cells, severe combined immunodeficiency mice engrafted with MG thymic lymphocytes were treated with anti‐Vβ5.1 antibody. Signs of pathogenicity (eg, acetylcholine receptor loss and complement deposits at the muscle end plates of chimeric mice) were prevented in anti‐Vβ5.1–treated severe combined immunodeficiency chimeras. Pathogenicity was mediated by autoantibodies against acetylcholine receptor. Thymic cells depleted of Vβ5.1‐positive cells in vitro before cell transfer were nonpathogenic, indicating that Vβ5.1‐positive cells are involved in the production of pathogenic autoantibodies. Acetylcholine receptor loss was prevented by Vβ5.1 targeting in HLA‐DR3 patients only, demonstrating specificity for HLA‐DR3–peptide complexes. The action of the anti‐Vβ5.1 antibody involved both the in vivo depletion of Vβ5.1‐expressing cells and an increase in the interferon‐γ/interleukin‐4 ratio, pointing to an immune deviation–based mechanism. This demonstration that a selective and specific T‐helper cell population is involved in controlling pathogenic autoantibodies in MG holds promise for the treatment of MG.

Synergistic induction of interleukin-6 production and gene expression in human thymic epithelial cells by LPS and cytokines☆

We examined the ability of LPS and several cytokines (TNF-alpha, IL-1-beta, IFN-gamma, IL-4) to modulate IL-6 production by cultured human thymic epithelial cells (TEC). IL-6 activity was measured by the hybridoma growth factor biological activity. Moderate but detectable IL-6 activity was spontaneously produced in the presence of serum proteins. LPS as well as the cytokines TNF-alpha and IL-1-beta was a potent inducer of IL-6, increasing, respectively, IL-6 levels by 9-, 28-, and 75-fold (mean values) while IL-4 and IFN-gamma provoked no significant effect. Interestingly, clearly different kinetics were observed for IL-6 induction by the various activation agents, the maximal effect being reached at 24, 48, and 72 hr, respectively for LPS, TNF-alpha, and IL-1-beta. Moreover, a synergistic effect of TNF-alpha and either LPS or IL-1-beta was observed. Indeed, TEC incubated with the cytokines in combination at optimal doses produced 5- to 170-fold more IL-6 than TEC stimulated with the cytokines individually. Neutralizing anti-IL-6 polyclonal and monoclonal antibodies completely blocked hybridoma proliferation stimulating activity of TEC supernatants; thus, implying that this activity is essentially due to IL-6. In situ hybridization analysis of cytocentrifuged TEC with an mRNA antisense probe specific for human IL-6 and labeled with 35S demonstrated that up to 90% of TEC could be induced to express the IL-6 gene. Computer-aided quantification of IL-6 mRNA levels indicated that upon stimulation with TNF-alpha combined to LPS, both the numbers of cells expressing IL-6 mRNA and the amounts of cytoplasmic IL-6 mRNA per cell were increased. Taken altogether these results demonstrate that LPS and/or cytokines can modulate and synergistically stimulate IL-6 production. In addition to a possible role in regulating normal thymic T cell activation, the IL-6 produced by TEC could be of pathophysiological relevance in disregulated situations such as in hyperplastic thymuses from patients with myasthenia gravis.

Evidence of enhanced recombinant interleukin-2 sensitivity in thymic lymphocytes from patients with myasthenia gravis: possible role in autoimmune pathogenesis.

We evaluated the activation state of thymic lymphocytes in patients with myasthenia gravis (MG) by cytofluorographic analysis of CD25 expression and by testing their sensitivity to recombinant interleukin-2 (rIL-2) in the absence of any known previous stimulation. We detected no phenotypic signs of activation in fresh MG thymic lymphocyte suspensions, while functional signs of activation were reflected in a significantly higher sensitivity to rIL-2 in MG patients than in controls. The responses to rIL-2 were time- and dose-dependent, were inhibited by a blocking anti-IL-2 receptor antibody, and were associated with an increase in CD25+ T cells in both patients and controls. The T cells with functional signs of previous activation may represent autoreactive cells involved in the autoimmune process and confirm thymus gland hyperactivity in MG. These cells could result from primary autosensitization against the thymic acetylcholine receptor (AChR)-like molecule or from altered migration of peripheral activated cells into an abnormal thymic environment. Our results also provide a clue for understanding the effect of thymectomy in myasthenia gravis.

T-cell antigenic sites involved in myasthenia gravis: correlations with antibody titre and disease severity.

We have evaluated the ability of eight synthetic peptides corresponding to selected regions of the alpha-subunit from human (H) or Torpedo (T) acetylcholine receptor (AChR) to stimulate proliferative responses of peripheral blood lymphocytes (PBL) and thymic cells from patients with Myasthenia Gravis (MG) in comparison to healthy controls. Using PBL, two of the peptides were most reactive: in the 40 myasthenic patients tested, peptide 169-181 (H) induced significant proliferative responses in 10 patients and peptide 351-368 (H) in five, while there was no response in any of the 34 healthy controls tested. Interestingly, clear associations between proliferation to peptides and clinical data were observed. Indeed, among responding patients, all presented thymic hyperplasia and most showed a high anti-AChR Ab titre and/or a severe form of the disease. In addition, responses to AChR cytoplasmic sequences were observed only in severely affected patients. Correlation with HLA-DR haplotype, sought in a subgroup of patients, indicated that response to 169-181 (H) is associated with HLA-DR5 in the patients presenting a high anti-AChR antibody titre. Using thymic lymphocytes, few responses were obtained with the human peptides, suggesting that the frequency of autoreactive cells is lower than in the blood. Similar to PBL, responses to peptides were observed only with lymphocytes isolated from hyperplastic thymuses. The correlations observed between responses to peptides and clinical parameters underline the pathophysiological relevance of our data and indicate that pathogenic and nonpathogenic T-cell antigenic sites involved in the anti-AChR response could be identified by this approach.

High recombinant interleukin-2 sensitivity of peripheral blood lymphocytes from patients with Myasthenia Gravis: Correlations with clinical parameters

Myasthenia Gravis (MG) is an autoimmune disorder of neuromuscular transmission associated with antibodies (Ab) against acetylcholine receptor (AChR). Autoantibody production is a T-cell-dependent phenomenon perhaps caused by aberrant immunoregulation. So far, a possible role for immunoregulatory molecules has not been investigated in the pathogenesis of MG. Since interleukin-2 (IL-2) is able to induce peripheral blood mononuclear cell (PBMC) proliferation without a previous activating signal and to upregulate IL-2-receptor expression, we have evaluated the activation state of PBMC in patients with MG, by cytofluorographic analysis of CD25 expression and by testing their sensitivity to recombinant IL-2 (rIL-2) without any known previous stimulation. We found no significant difference in CD25 expression in a large group of patients compared to controls. However, proliferative responses to rIL-2 were significantly higher in MG patients than in controls. In MG, as in controls, this response was time- and dose-dependent, was inhibited by an anti-IL-2 receptor Ab and correlated with an increased percentage of CD25+ T cells after rIL-2 exposure. The response was greater in patients with a high anti-AChR Ab titer and a severe form of the disease, and in patients tested before thymectomy. Thus blood T cells in MG showed functional signs of preactivation (high sensitivity to rIL-2 alone) without detectable CD25 expression on fresh cells, raising the possibility of aberrant IL-2 receptor regulation and/or expression in MG T cells. Decreased sensitivity to rIL-2 after thymectomy, associated with general clinical improvement, suggests a role for activated cells originating from the thymus in the pathogenesis of MG, and is of clinical relevance in patient follow-up. Our findings also provide a new approach in the study of MG pathogenesis: the search for aberrant immunoregulation mechanisms linked to defects in lymphokine circuits.

Antibodies to thymic epithelial cells in myasthenia gravis

The presence of anti-thymus antibodies was investigated in the serum of 36 patients with myasthenia gravis (MG). Using an immunofluorescence technique on frozen thymic sections, we found 45% of patients sera reacting with normal or MG thymuses. Staining was confined to subcapsular and medullary keratin-positive epithelial cells. Thirty-five out of 36 sera from healthy controls and all 15 sera from patients presenting another autoimmune disorder were negative. Antibodies to thymic epithelial cells were almost exclusively detected in patients presenting thymic hyperplasia and did not disappear after thymectomy. They were not clearly associated with antiacetylcholine receptor antibody titer, nor with disease severity. Their strong association to thymic abnormalities highlights the role of the thymus in pathogenesis of MG. The reasons for the appearance of these antibodies, the structure they recognize on thymic epithelial cells and their possible etiological role are discussed.

Prospects for a T-cell receptor vaccination against myasthenia gravis.

T-cell receptor (TCR) vaccination has been proposed as a specific therapy against autoimmune diseases. It is already used in clinical trials, which are supported by pharmaceutical companies for the treatment of multiple sclerosis, rheumatoid arthritis and psoriasis. Current vaccine developments are focusing on enhancement of immunogenicity as well as selecting the best route of immunization and adjuvant to favor the therapeutic effect. In the meantime, academic laboratories are tackling the regulatory mechanisms involved in the beneficial effect of the vaccines to further understand how to control the therapeutic tool. Indeed, several examples in experimental models of autoimmune diseases indicate that any specific therapy may rely on a delicate balance between the pathogenic and regulatory mechanisms. This review presents a critical analysis of the potential of such therapy in myasthenia gravis, a prototype antibody-mediated disease. Indeed, a specific pathogenic T-cell target population and a TCR-specific regulatory mechanism mediated by anti-TCR antibodies and involved in protection from the disease have recently been identified in a patient subgroup. The presence of spontaneous anti-TCR antibodies directed against the pathogenic T-cells that may be boosted by a TCR vaccine provides a rationale for such therapy in myasthenia gravis. The development of this vaccine may well benefit from experience gained in the other autoimmune diseases in which clinical trials are ongoing.

Follow-up of soluble interleukin-2 receptor levels after thymectomy in patients with myasthenia gravis

Soluble interleukin-2 receptor (sIL-2R) levels were followed up after thymectomy by a quantitative immunoradiometric assay in 59 patients with myasthenia gravis (MG). Increased levels of sIL-2R were found in 30.5% of the patients before thymectomy. Serum levels were significantly higher in severely affected patients. Sequential sampling after thymectomy indicated a significant and progressive decline of sIL-2R levels within 2 years after surgery, which was well associated with clinical improvement or remission. The sIL-2R purified from sera of patients with MG had a molecular mass of 45 kDa as the normal sIL-2R. The decline after thymectomy of sIL-2R titers suggests a possible role of the thymus in the occurrence of sIL-2R in the periphery. Soluble IL-2R levels may represent a marker of disease severity in MG, which might be useful in the follow-up of individual patients.

In vitro interleukin-1 (IL-1) production in thymic hyperplasia and thymoma from patients with myasthenia gravis

Using a biological test, we measuredin vitro thymic epithelial cell IL-1 activity of 14 thymomas and 8 hyperplasia from myasthenic patients. Our results demonstrate that thymic epithelial cells from hyperplastic thymuses spontaneously produce high amounts of IL-1 compared to controls, while cells from thymomas produce very low amounts of IL-1. After LPS stimulation the difference between patients and controls is no longer significant. Immunohistochemistry studies demonstrate a limited number of IL-1-positive cells on sections from normal thymuses and a variable number of IL-1-positive cells on diseased thymuses, not clearly correlated to thein vitro production. In hyperplastic thymuses an association is found between the level of hyperplasia andin vitro IL-1 production, suggesting a role for IL-1 in the expansion of thymic lymphoid follicles. In thymoma, the low IL-1 production and the loss of corticomedullary organisation raise the possibility that some autoreactive clones could emerge from the lymphocyte pool, present in the abnormal tumoral microenvironment.