Frequency of IgG⁺ memory cells specific for GAD65 (A) and TT (B) was analyzed by limiting dilution assays at week 0 and 36 for twin A, and for three healthy donors (HD).

Binding of IgG from twin A (A) and twin B (B) isolated at t = 0 and at t = 36 weeks to radiolabeled GAD65 was analyzed in the presence of recombinant Fab derived from GAD65-specific monoclonal antibodies. Grey bars represent competitive binding of healthy control IgG. DPA, b96.11, MICA-3, DPD, N-GAD65mAb, and b78 are monoclonal antibodies specific for GAD65. The control Fab does not bind GAD65.

(A) The mean frequency of VH usage for each isotype was determined by quantitative real time PCR analysis on B cells from patients at week 0 and at week 36. As control VH usage by B cells from a healthy donor (HD) is shown. (B) CDR3 length distribution in IgM⁺, IgG⁺ and IgA⁺ B cell of twin A and B before and after rituximab treatment was analyzed by spectratyping[30]. The x axis of spectratype indicates CDR3 length, whereas the y axis represents the frequency of CDR3 regions. The percentage of VH utilization among all three isotypes is indicated on each profile. Arrows indicate overrepresented CDR3 regions.

Rituximab treatment induces a 2-fold decrease of GAD65 specific serum antibody titer. (A) Serum samples from twin A and twin B were collected before and 8, 16, 36 weeks after rituximab treatment, and GAD65 specific antibody titers were measured by ELISA. The titers of 8 healthy donors ranged between undetectable and 1/80. (B) Inhibition of GAD65 enzyme activity was tested with IgG of twin A and twin B isolated at week 0 and 36 after starting the anti-CD20 treatment. (C,D) TT and Rotavirus antibody titers. Serum from twin A and twin B was collected at week 0 and 36 post rituximab treatment, and antibody titer was measured by ELISA. Eight healthy donors (HD) were used as controls. (E, F) Pneumococcal polysaccharide (PnP) antigens antibody titers in twin A (E) and in twin B serum (F), at week 0 and 36 post treatment, x axes show the different PnP antigens tested.

(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.

(A) Sections were stained with a reference anti-GAD65 monoclonal antibody, column 1, and IgG of twin B, column 2. Column 3 represents the merged images and anti-calbindin staining (blue). Row a: mouse cerebellum, Purkinje cell layer, t = 0. Row b: mouse cerebellum, Purkinje cell layer, t = 36 weeks after rituximab treatment. Row c: healthy human cerebellum, Purkinje cell layer, t = 0. Row d: healthy human cerebellum, Purkinje cell layer, t =  36 weeks after rituximab treatment. Solid arrows mark GAD positive inhibitory synapses stained with IgG from twin B, thin arrows show presynaptic terminals staining only with the anti-GAD65 monoclonal antibody. Slides were scanned at 1024×1024 pixel resolution with a Leica TCS NT confocal microscope, three channel recordings were performed consecutively in Z-series with gaps of 250 nm between the optical slices. Parameters were held constant between recordings of different samples. The stainings are representative for analyses performed with sections from 3 mouse and 3 healthy human samples with immunoglobulin derived both from twin A and B. (B) Differential recognition of brain antigen before and after rituximab treatment. Serial consecutive sections of three mouse brains were stained with IgG of twin B from t = 0, twin B IgG week 36. Three fields of four different brain areas were analyzed per mouse. Fluorescence signal intensities were examined by 10 linear measurements for a total of 600 µm per brain area and corrected for background staining. For each the ratio between twin B IgG fluorescence intensity and anti-GAD65 monoclonal antibody fluorescence intensity was calculated. Data represented are subtracted from the HD signals in the specific areas. Significant differences in staining were determined with unpaired two-tailed T test (*** <0.0001; ** <0.001; * <0.05).