Table 1.
SS patients analysed in this study.
Figure 1.
Isolation strategy of single naïve B cells and comparison of the frequencies of naïve, memory switched and unswitched B cells in SS patients and HD.
(A) PBMC from SS patients were surface labeled with fluorochrome-coupled anti-CD19, anti-CD3, anti-IgD and anti-CD27. CD19+CD3− cells were gated and analyzed for IgD and CD27 expression. (B) The frequencies of peripheral naïve (CD3−CD19+CD27−IgD+), memory switched (CD3−CD19+CD27+IgD–) and unswitched (CD3−CD19+CD27+IgD+) B cells among all CD19+ B cells are shown. Differences between patients and HD were statistically significant using the nonparametric Mann-Whitney U test (p value is reported over each graph). Error bars indicate standard error of the mean (SEM) for individual patient or control.
Figure 2.
Ig gene analysis of naïve and memory B cells from SS patients and HD.
Single naïve and memory unswitched and switched B cell antibodies from SS patients and HD [19]–[21] were analyzed for (A) Ig VH family and JH gene usage, and (B) IgH CDR3 aa length and number of positive charges. The absolute number of sequences analyzed is reported over the graph. Error bars in bar graphs indicate standard error of mean (SEM) for individual patient or control.
Figure 3.
Ig gene analysis of naïve and memory B cells from SS patients and HD.
Single naïve and memory unswitched and switched B cell antibodies from SS patients and HD [19]–[21] were analyzed for Ig Vκ and Jκ (A) and Ig Vλ and Jλ (B) gene usage. The absolute number of sequences analyzed is reported over each graph. Error bars in bar graphs indicate standard error of mean (SEM) for individual patient or control.
Figure 4.
Interference of antigen selection in memory B cells from SS patients.
(A) Replacement:silent ratio (R/S ratios) were calculated for the complementary determining regions CDRs (black) and framework regions (white) for the heavy chain of both memory unswitched and switched B cells. A significant higher R/S ratio in the CDRs was observed in memory switched vs memory unswitched B cells. (B) The graphs show the ratio of replacement mutations in CDR1 and CDR2 (RCDR) to the total number of mutations in V region (MV) plotted against MV for the memory unswitched and switched B cells from SS patients. The dark and the light grey area indicate the 90% and 95% confidence limits for the probability of random mutations, respectively. A data point outside these areas represents a sequence that was antigen selected. The data were obtained using the Immunoglobulin Analysis Tool software [22].
Figure 5.
Polyreactivity of naïve and memory B cell antibodies from SS patients and HD.
Naïve B cell antibodies from SS patients (n = 60), HD (n = 41) and memory B cells from SS patients (n = 39) were tested for reactivity with dsDNA (top left), ssDNA (top right), LPS (bottom left) and insulin (bottom right) by ELISA. Each graph shows the reactivity at a concentration of 1 µg/ml and it shows the result of two independent experiments. The cut-off OD (450 nm) at which antibodies were considered reactive is shown by the horizontal lines. Data points represent individual antibodies. Internal controls for polyreactivity (star dots) are shown in each graph and include mGO53 (negative; [17]), JB40 (low polyreactive; [17]), and ED38 (highly polyreactive; [17]).
Figure 6.
Hep-2 cell IFA self-reactivity of naïve and memory B cell antibodies from SS patients and HD.
Naïve and memory B cell antibodies from SS patients and HD (naïve B cells) were tested for self-reactivity by Hep-2 cell IFA assay. (A) Examples of cytoplasmic, cytoplasmic and nuclear, and nuclear Hep-2 cell staining pattern are shown. (B) Graph bars summarize the frequency of Hep-2 cell reactive antibodies with cytoplasmic, nuclear and cytoplasmic, nuclear reactivity, and non Hep-2 cell reactive antibodies in SS patients (naïve and memory B cell antibodies) and HD (naïve B cell antibodies). P value compares ANA+ (cytoplasmic only and nuclear ± cytoplasmic reactive) versus ANA– (non Hep-2 reactive) naïve clones from SS patients and HD. The percentage of self-reactivity for each population is reported over each graph. The number of tested antibodies is indicated below each graph.