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Long-lived plasma cells and memory B cells produce pathogenic anti-GAD65 autoantibodies in Stiff Person Syndrome.

Rizzi M, Knoth R, Hampe CS, Lorenz P, Gougeon ML, Lemercier B, Venhoff N, Ferrera F, Salzer U, Thiesen HJ, Peter HH, Walker UA, Eibel H - PLoS ONE (2010)

Bottom Line: Studying the B cell compartment and the anti-GAD65 B cell response in two monozygotic twins suffering from SPS, who were treated with the B cell-depleting monoclonal anti-CD20 antibody rituximab, we found that the humoral autoimmune response in SPS is composed of a rituximab-sensitive part that is rapidly cleared after treatment, and a rituximab-resistant component, which persists and acts as a reservoir for autoantibodies inhibiting GAD65 enzyme activity.Both subsets represent only a fraction of anti-GAD65 autoantibody secreting cells.Therefore, the identification and targeting of this compartment is a key factor for successful treatment planning of SPS and of similar autoimmune diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Rheumatology and Clinical Immunology, University Medical Center Freiburg, Freiburg, Germany.

ABSTRACT
Stiff person syndrome (SPS) is a rare, neurological disorder characterized by sudden cramps and spasms. High titers of enzyme-inhibiting IgG autoantibodies against the 65 kD isoform of glutamic acid decarboxylase (GAD65) are a hallmark of SPS, implicating an autoimmune component in the pathology of the syndrome. Studying the B cell compartment and the anti-GAD65 B cell response in two monozygotic twins suffering from SPS, who were treated with the B cell-depleting monoclonal anti-CD20 antibody rituximab, we found that the humoral autoimmune response in SPS is composed of a rituximab-sensitive part that is rapidly cleared after treatment, and a rituximab-resistant component, which persists and acts as a reservoir for autoantibodies inhibiting GAD65 enzyme activity. Our data show that these potentially pathogenic anti-GAD65 autoantibodies are secreted by long-lived plasma cells, which may either be persistent or develop from rituximab-resistant memory B lymphocytes. Both subsets represent only a fraction of anti-GAD65 autoantibody secreting cells. Therefore, the identification and targeting of this compartment is a key factor for successful treatment planning of SPS and of similar autoimmune diseases.

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Antibodies recognizing linear GA65 epitopes are sensitive to rituximab treatment.(A–C) Sera isolated from patients before (w0: week 0) and after rituximab treatment (w36: week 36) and from 2 healthy donors (HD1, 2) were hybridized to peptide arrays covering the entire GAD65 amino acid sequence. In graph A–C are represented the peptides specifically recognized by SPS patients sera. Filled and open symbols represent samples from patients before and after B cells depletion, respectively. Significant differences in binding were determined by one-tailed T-test (* <0.1; ** <0.05). For all peptides with the exception of 79–93 (PCSCSKVDNNYAFLH), p-values for the means between twin A/B week 0 and the healthy donor sera were <0.05. Data are representative of triplicate wells of 3 independent experiments. (D–H) On the same peptide array were hybridized sera from twin A collected before and 8, 16, 36 weeks after rituximab treatment. Graphs represent relevant peptides. Data are representative of triplicate wells of 2 independent experiments.
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pone-0010838-g005: Antibodies recognizing linear GA65 epitopes are sensitive to rituximab treatment.(A–C) Sera isolated from patients before (w0: week 0) and after rituximab treatment (w36: week 36) and from 2 healthy donors (HD1, 2) were hybridized to peptide arrays covering the entire GAD65 amino acid sequence. In graph A–C are represented the peptides specifically recognized by SPS patients sera. Filled and open symbols represent samples from patients before and after B cells depletion, respectively. Significant differences in binding were determined by one-tailed T-test (* <0.1; ** <0.05). For all peptides with the exception of 79–93 (PCSCSKVDNNYAFLH), p-values for the means between twin A/B week 0 and the healthy donor sera were <0.05. Data are representative of triplicate wells of 3 independent experiments. (D–H) On the same peptide array were hybridized sera from twin A collected before and 8, 16, 36 weeks after rituximab treatment. Graphs represent relevant peptides. Data are representative of triplicate wells of 2 independent experiments.

Mentions: Since we found that autoantibodies to conformational epitopes of GAD65 were still present after rituximab treatment, we also compared the specificities against linear epitopes before and after rituximab therapy and analyzed binding of serum IgG from both patients to 96 overlapping linear 15-mer peptides spanning the entire GAD65 amino acid sequence. The peptide array analysis revealed five peptides located within the first 99 amino acid residues of GAD65, which were recognized by the IgG of both patients before rituximab treatment was initiated (Fig. 5 A–C and Fig. S1). In addition, a peptide spanning residues 14–28 was detected by IgG from twin A but not from twin B (Fig. S1). Anti-CD20 treatment changed the binding of IgG of twin A to 5 peptides spanning residues 1–15, 37–57, and 79–99, whereas the peptide recognition pattern of twin B remained constant (Fig. 5 A–C). Lower IgG titers for these 5 peptides were found already 8 weeks after rituximab treatment and progressively decreased later on (Fig. 5 D–H). Since the half-life of IgG is 3–5 weeks [36], these data indicate that the B cells producing these specificities are eliminated by treatment and the antibodies are progressively diluted in the serum.


Long-lived plasma cells and memory B cells produce pathogenic anti-GAD65 autoantibodies in Stiff Person Syndrome.

Rizzi M, Knoth R, Hampe CS, Lorenz P, Gougeon ML, Lemercier B, Venhoff N, Ferrera F, Salzer U, Thiesen HJ, Peter HH, Walker UA, Eibel H - PLoS ONE (2010)

Antibodies recognizing linear GA65 epitopes are sensitive to rituximab treatment.(A–C) Sera isolated from patients before (w0: week 0) and after rituximab treatment (w36: week 36) and from 2 healthy donors (HD1, 2) were hybridized to peptide arrays covering the entire GAD65 amino acid sequence. In graph A–C are represented the peptides specifically recognized by SPS patients sera. Filled and open symbols represent samples from patients before and after B cells depletion, respectively. Significant differences in binding were determined by one-tailed T-test (* <0.1; ** <0.05). For all peptides with the exception of 79–93 (PCSCSKVDNNYAFLH), p-values for the means between twin A/B week 0 and the healthy donor sera were <0.05. Data are representative of triplicate wells of 3 independent experiments. (D–H) On the same peptide array were hybridized sera from twin A collected before and 8, 16, 36 weeks after rituximab treatment. Graphs represent relevant peptides. Data are representative of triplicate wells of 2 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2877104&req=5

pone-0010838-g005: Antibodies recognizing linear GA65 epitopes are sensitive to rituximab treatment.(A–C) Sera isolated from patients before (w0: week 0) and after rituximab treatment (w36: week 36) and from 2 healthy donors (HD1, 2) were hybridized to peptide arrays covering the entire GAD65 amino acid sequence. In graph A–C are represented the peptides specifically recognized by SPS patients sera. Filled and open symbols represent samples from patients before and after B cells depletion, respectively. Significant differences in binding were determined by one-tailed T-test (* <0.1; ** <0.05). For all peptides with the exception of 79–93 (PCSCSKVDNNYAFLH), p-values for the means between twin A/B week 0 and the healthy donor sera were <0.05. Data are representative of triplicate wells of 3 independent experiments. (D–H) On the same peptide array were hybridized sera from twin A collected before and 8, 16, 36 weeks after rituximab treatment. Graphs represent relevant peptides. Data are representative of triplicate wells of 2 independent experiments.
Mentions: Since we found that autoantibodies to conformational epitopes of GAD65 were still present after rituximab treatment, we also compared the specificities against linear epitopes before and after rituximab therapy and analyzed binding of serum IgG from both patients to 96 overlapping linear 15-mer peptides spanning the entire GAD65 amino acid sequence. The peptide array analysis revealed five peptides located within the first 99 amino acid residues of GAD65, which were recognized by the IgG of both patients before rituximab treatment was initiated (Fig. 5 A–C and Fig. S1). In addition, a peptide spanning residues 14–28 was detected by IgG from twin A but not from twin B (Fig. S1). Anti-CD20 treatment changed the binding of IgG of twin A to 5 peptides spanning residues 1–15, 37–57, and 79–99, whereas the peptide recognition pattern of twin B remained constant (Fig. 5 A–C). Lower IgG titers for these 5 peptides were found already 8 weeks after rituximab treatment and progressively decreased later on (Fig. 5 D–H). Since the half-life of IgG is 3–5 weeks [36], these data indicate that the B cells producing these specificities are eliminated by treatment and the antibodies are progressively diluted in the serum.

Bottom Line: Studying the B cell compartment and the anti-GAD65 B cell response in two monozygotic twins suffering from SPS, who were treated with the B cell-depleting monoclonal anti-CD20 antibody rituximab, we found that the humoral autoimmune response in SPS is composed of a rituximab-sensitive part that is rapidly cleared after treatment, and a rituximab-resistant component, which persists and acts as a reservoir for autoantibodies inhibiting GAD65 enzyme activity.Both subsets represent only a fraction of anti-GAD65 autoantibody secreting cells.Therefore, the identification and targeting of this compartment is a key factor for successful treatment planning of SPS and of similar autoimmune diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Rheumatology and Clinical Immunology, University Medical Center Freiburg, Freiburg, Germany.

ABSTRACT
Stiff person syndrome (SPS) is a rare, neurological disorder characterized by sudden cramps and spasms. High titers of enzyme-inhibiting IgG autoantibodies against the 65 kD isoform of glutamic acid decarboxylase (GAD65) are a hallmark of SPS, implicating an autoimmune component in the pathology of the syndrome. Studying the B cell compartment and the anti-GAD65 B cell response in two monozygotic twins suffering from SPS, who were treated with the B cell-depleting monoclonal anti-CD20 antibody rituximab, we found that the humoral autoimmune response in SPS is composed of a rituximab-sensitive part that is rapidly cleared after treatment, and a rituximab-resistant component, which persists and acts as a reservoir for autoantibodies inhibiting GAD65 enzyme activity. Our data show that these potentially pathogenic anti-GAD65 autoantibodies are secreted by long-lived plasma cells, which may either be persistent or develop from rituximab-resistant memory B lymphocytes. Both subsets represent only a fraction of anti-GAD65 autoantibody secreting cells. Therefore, the identification and targeting of this compartment is a key factor for successful treatment planning of SPS and of similar autoimmune diseases.

Show MeSH
Related in: MedlinePlus