Jo-1 autoantigen-specific B cells are skewed towards distinct functional B cell subsets in anti-synthetase syndrome patients

Figures

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  Fig. 1. Jo-1-specific antibody is detected in sera and stimulated PBMC cultures from subjects with Jo-1 ARS. Serum and PBMCs were collected from individuals with clinical laboratory-confirmed cases of Jo-1 ARS disease (n = 9), IIM patients with “Other Ab” (anti-PL-7, anti-EJ with anti-Ro52, anti-Mi-2, and anti-TIF1- γ), and healthy subjects (n = 8). a ELISA was used to measure serum IgG (left) or IgM (right) antibody bound to purified GST-Jo-1 fusion protein (black) and purified GST protein (gray). b ELISA was used to measure serum IgG (left) or IgM (right) antibody bound to GST-Jo-1 in the absence (black) or presence of excess soluble GST-Jo-1 competitor (gray) to detect Jo-1-specific autoantibody. c, d PBMCs collected from n = 5 Jo-1+ subjects were separated into individual wells and polyclonally stimulated as in the “Methods” section to drive B cell differentiation into antibody-secreting cells. The fraction of positive wells is shown secreting c total antibody or d anti-Jo-1. d Results from two representative patients are shown. Dashed line defines positive wells with a mean fluorescence that is 3 standard deviations above the mean O.D. of all wells

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  Fig. 2. JBCs are detected among IgM+ and IgG+ B cells, with the majority being non-class-switched. PBMCs from healthy controls (top) or Jo-1 ARS (bottom) patients were stained and analyzed using flow cytometry. a Live, CD19+ CD5− CD14− CD16− cells were gated and anti-GST secondary antibody discriminated between Jo-1/GST-binding (Jo-1+) and non-JBCs (Jo-1−) B cells. b Cells were further gated on IgM versus IgG expression by non-JBC and JBC. Representative plots from healthy control and Jo-1 ARS donors are shown. c–f Flow cytometry identifies the indicated populations among n = 5 stable Jo-1 ARS (triangles), n = 5 active Jo-1 ARS (circles), and n = 8 healthy controls (diamonds). c JBCs were identified as in panel a and their frequency of total B cells was determined. d The IgM+ or IgG+ cell frequency (gated as in panel b) of JBCs (gated as in panel a) is shown for Jo-1 ARS patients that had > 20 JBC events (n = 6). e The frequency of JBCs within IgM+ (e) or IgG+ (f) B cells is shown. Individual donors are plotted, and bars represent the mean ± SD. p values were determined using the Mann-Whitney U test, and are indicated on each panel

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  Fig. 3. JBCs are enriched among CD21lo, but not BND B cell subsets. PBMCs from healthy controls or Jo-1 ARS patients were stained and analyzed using flow cytometry. Live B cells were identified as in Fig. 2 and a BND (IgMlo IgD− CD27−) or b CD21lo B cells were identified; representative plots are shown for healthy controls (top) or Jo-1 ARS patients (bottom). c–e n = 5 stable Jo-1 ARS (triangles), n = 5 active Jo-1 ARS (circles), and n = 8 healthy controls (diamonds). Only those Jo-1 ARS patients that had > 20 JBC events (n=6) were included for JBC sub-analysis. c The frequency of BND B cells among JBCs and non-JBCs in Jo-1 ARS patients (identified as in Fig. 2) or from healthy donors is shown. d, e CD21lo B cells were further gated on d IgM+ or e IgG+ cells and the frequencies of the indicated subsets are shown. Individual donors are plotted, and bars represent the mean ± SD. p values were determined using the Mann-Whitney U test, and significant values are indicated on each panel

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  Fig. 4. JBCs are enriched among CD21lo IgM+ memory cells. PBMCs from healthy controls (top) or Jo-1 ARS (bottom) patients were stained and analyzed based on IgD, IgM, IgG, and CD27 expression using flow cytometry. Live JBCs and non-JBCs (Jo-1 ARS) or total B cells (healthy donors) were identified as in Fig. 2. a, b Representative plots are shown for healthy control (top) or Jo-1 ARS (bottom) donors for the indicated markers. IgM and IgG expression was examined among CD27+ IgD− cells gated as in panel a. c–h The indicated phenotypic subsets were examined in n = 5 stable Jo-1 ARS (triangles), n = 5 active Jo-1 ARS (circles), and n = 8 healthy controls (diamonds). Only those Jo-1 ARS patients that had > 20 JBC events were included for JBC sub-analysis of either CD21lo (c–e) or all cells (f–h) for c, f IgD+ CD27+ IgM+, d, g IgD− CD27+ IgM+, and e, h IgD− CD27+ IgG+ subsets. Individual donors are plotted, and bars represent the mean ± SD. p values were determined using the Mann-Whitney U test, and significant values are indicated on each panel

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  Fig. 5. JBCs are not increased among transitional B cells or plasmablasts. PBMCs from healthy controls (top) or Jo-1 ARS (bottom) patients were stained and transitional B cells (CD24hi CD38hi CD27− IgD+ CD10+) or plasmablasts (CD24− CD38high) were identified using flow cytometry. a, b Representative plots are shown for healthy control (top) or Jo-1 ARS (bottom) donors for the indicated markers. b CD24hi CD38hi cells as gated in panel a are further gated on IgD/CD27 (left) and the resulting IgD+ CD27− cells are gated on CD10 (right). c, d The indicated phenotypic subsets were examined in n = 5 stable Jo-1 ARS (triangles), n = 5 active Jo-1 ARS (circles), and n = 8 healthy controls (diamonds). Only those Jo-1 ARS patients that had > 20 JBC events were included for JBC sub-analysis of c plasmablasts or d transitional B cells. Individual donors are plotted, and bars represent the mean ± SD. p values were determined using the Mann-Whitney U test, and significant values are indicated on each panel

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  Fig. 6. JBCs are identified following stimulation in vitro but are predominantly IgM+ (non-class-switched). PBMCs from Jo-1 ARS patients with active disease were stimulated as described in the “Methods” section. Cultured cells from 40 to 60 wells per patient were harvested on day 6 and stained for flow cytometry analysis. a Representative pseudocolor plots describing the gating strategy are shown for the selection of live, CD19+ IgM+/− Jo-1 binding populations. The frequencies for each of these plots are indicated. b Cells were gated on live, CD19+ JBCs and the mean frequency of IgM+ and IgM− cells was calculated per donor; individual donors are plotted. c Pseudocolor plot depicting the CD21lo B cells in JBCs and non-JBCs. d The mean frequency of CD21lo B cells within the indicated populations was calculated for per donor and individual donors are plotted. Statistical significance between populations as calculated using the Mann-Whitney U test is indicated. Bars indicate the mean ± SD

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  Fig. 7. A lower frequency of JBCs acquire a plasmablast phenotype following stimulation in vitro compared to non-JBCs from the same Jo-1 ARS patients. PBMCs from n = 5 Jo-1 ARS patients with active disease were polyclonally stimulated as in the “Methods” section. Cultured cells from n=40-60 wells per patient were harvested on day 6 and stained for flow cytometry analysis. a CD24 and CD38 expression is shown in representative flow plots derived from a single well of stimulated PBMCs. Comparison of mean frequencies of b CD38hi CD24− B lymphocytes or c CD38hi CD24mid B lymphocytes between JBC and non-JBC populations. Significant differences between IgM+ JBC and IgM+ non-JBC or IgM− JBC and IgM− non-JBC populations as calculated using Mann-Whitney U test are indicated. The data are represented as the mean for each population within a given stimulation, and the bars indicate the mean ± SD