Fig 1: IgA1 in Sm/RNP immune complexes enhances IFNα production by human pDCsAll pDCs were enriched from PBMCs freshly isolated from healthy control (HC) donor blood. All immune complexes (ICs) were generated with Sm/RNP as the RNA-containing nuclear antigen (A) pDC IFNα secretion after incubation with Sm/RNPs alone or with Sm/RNP ICs generated with SLE serum, SLE serum ΔIgA1 or SLE IgA1. Each symbol shape representing a different pDC donor, mean and range shown. (B) Paired analysis of pDC IFNα secretion after incubation with Sm/RNP ICs generated with either SLE serum or SLE serum ΔIgA1 from 8 SLE serum donors. (C) Paired analysis of pDC IFNα secretion with the same SLE serum donor and HC pDC donor assessed at 4 times over 14 months. (D) Paired analysis of pDC IFNα2 (left) and TNF (right) secretion after incubation with SLE serum or SLE serum ΔIgA1 ICs. (E) pDC IFNα secretion after pDCs were incubated with PBS, mIgG1 isotype control, anti-FcγRIIa, anti-FcαR, aggregated IgG or aggregated IgA for 2 hours prior to the addition ICs generated from SLE serum. Data are shown as a percent of PBS control with mean and range (raw data shown in Supplemental Figure 4E). (A) One way ANOVA, repeated measures, Bonferroni correction for multiple comparisons between all groups. Only significant comparisons are shown (*** p<0.001 and ** p<0.01). (B-D) Ratio paired t-test (** p<0.01 and * p<0.05).(E) One way ANOVA, repeated measures, Bonferroni correction for comparing all blocking conditions to the isotype control (*** p<0.001). SLE serum donors; n=1 (A, C and E), n=4 (D), n=8 (B). HC pDC donors; n=1 (B, C and D), n=3 (E), n=4 (A).
Fig 2: IgA1 autoantibodies contribute to IC association with pDCsAll pDCs were enriched from PBMCs freshly isolated from healthy control (HC) donor blood and ICs were generated with AlexaFluor647-labeled Sm/RNPs (Sm/RNP-AF647). (A) pDCs were incubated with Sm/RNP-AF647 alone (bottom row) or Sm/RNP-AF647 ICs generated from SLE serum (top row) or SLE serum ΔIgA1 (middle row), for 3–20 hours. Representative flow cytometry plots show the percent of Sm/RNP-AF647+ pDCs. The Sm/RNP-AF647+ gate was determined by selecting where binding of Sm/RNP-AF647 control was approximately 1% or less (bottom row). (B) Percent IC+ pDCs at 12 hours after incubation with ICs generated with SLE serum or SLE serum ΔIgA1 from 4 SLE donors. (C) Correlation between IC+ pDCs (%) at 12 hrs and IFNα secretion at 20 hours (ng/mL) after incubation with Sm/RNP-AF647 ICs generated from SLE serum (solid lavender symbols) or SLE serum ΔIgA1 (open blue symbols). Plot shows the same IFNα secretion data from (B) with each symbol shape corresponding to the SLE serum donor. (D) Correlation of 12 hr IC+ pDCs (%) and normalized 20 hr IFNα production (% max) for combined experiments in which pDCs were incubated with Sm/RNP-AF647 ICs generated with SLE serum or SLE serum ΔIgA1. (B) Ratio paired t-test (* p<0.05) (C, D) r = Pearson’s correlation coefficient. SLE serum donors; n=1(A), n=4 (B, C, D). HC pDC donors; n=1 (A, B, C), n=4 (D).
Fig 3: RNA-containing immune complexes generated from SLE donors with anti-Sm/RNP antibodies(A) Schematic showing reagents used to generate immune complexes (ICs) that contained both IgG and IgA (I.), mainly IgG (II.) or only IgA1 (III.) from SLE donors with IgG and IgA anti-Sm/RNP antibodies. IgA1 depleted serum (SLE serum ΔIgA1) and purified SLE IgA1 were generated from SLE serum by using an IgA1 binding substrate column and collecting the flow through and eluted protein, respectively. ICs were made by mixing the different antibody reagents with RNA-containing Sm/RNPs. (B) Levels of IgA anti-Sm/RNP (left) and IgG anti-Sm/RNP (right) antibodies in SLE serum (purple circles), SLE serum ΔIgA1 (blue open squares) and SLE IgA1 (red diamonds), as measured by ELISA. Representative data for one serum donor is shown.
Fig 4: IgA1 autoantibodies enhance pDC internalization of immune complexesEnriched pDCs from healthy individuals were incubated with Sm/RNP-AF647 alone or Sm/RNP-AF647 ICs generated with either SLE serum or SLE serum ΔIgA1 for 12 hrs. (A) Representative confocal images of the three categories of staining observed with DAPI (blue), CD123 (green) and Sm/RNP-AF647 ICs (magenta). Group I (left), pDCs that had no ICs bound or internalized or very faint IC signaling indicating low level or diffuse ICs bound. Group II (middle), pDCs with either small (top cell) or large (bottom cell) internalized ICs. Group III (right), pDCs with either small (top cell) or large (bottom cell) ICs that were bound but not internalized. Images shown are a composite of collapsed z-stacks, Supplemental Fig. 6 shows examples with full z-stacks to demonstrate the difference between internalized and bound ICs. (B) Donut plots show the proportion of pDCs in each group for each experimental condition: Sm/RNP-AF647 with no serum (left), Sm/RNP-AF647 ICs generated with SLE serum (middle), and Sm/RNP-AF647 ICs generated with SLE serum ΔIgA1 (right). The center of the donut plot shows the number of cells imaged and analyzed in each group. Chi-squared analysis performed between all groups and statistically significant comparisons shown (** p<0.01, *** p<0.001).
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