Circulating Pentraxin3-Specific B Cells Are Decreased in Lupus Nephritis

Abstract

Background: Pentraxin3 (PTX3) is overexpressed in kidneys of patients developing lupus nephritis (LN). Active LN is associated with reduced anti-PTX3 antibodies. However, abnormalities of B cell differentiation against PTX3 have not been characterized in systemic lupus erythematosus (SLE). Objective: Characterization of PTX3-specific (PTX3⁺) B cells in peripheral blood of SLE patients with or without LN and healthy donors (HD). Patients and Methods: SLE patients without LN, biopsy-proven LN and matched HD were analyzed. Active LN was defined as proteinuria>0.5 g/day or CrCl<60 ml/min/1.73 m² with active urinary sediment. Peripheral B cells were analyzed for direct PTX3 binding by flow cytometry using PTX3 labeled with cyanine 5 (Cy5) and phycoerythrin (PE). Results: Initially, a flow cytometry based assay to identify PTX3⁺ B cells was developed by demonstrating simultaneous binding of PTX3-Cy5 and PTX3-PE. Specificity of B cells was validated by blocking experiments using unlabeled PTX3. We could identify circulating PTX3⁺ B-cells in HD and patients. Notably, LN patients showed a significantly diminished number of PTX3⁺ B cells (SLE vs. LN p = 0.033; HD vs. LN p = 0.008). This decrease was identified in naïve and memory B cell compartments (naïve: SLE vs. LN p = 0.028; HD vs. LN p = 0.0001; memory: SLE vs. LN p = 0.038, HD vs. LN p = 0.011). Conclusions: Decreased PTX3⁺ B cells in LN within the naïve and memory compartment suggest their negative selection at early stages of B cell development potentially related to a decreased regulatory function. PTX3⁺ B cells could candidate for autoantigen-defined regulatory B cells as a striking abnormality of LN patients.

Keywords: PTX3+ B cells; SLE; biomarkers; flow-cytometry; lupus nephritis.

Figure 1

Identification of PTX3+ B cells among CD19+CD20+ B cells. (A) Three representative dot plots of the PTX3-specific B cells before and after blocking with unlabeled PTX3. Only B cells staining positive for both PTX3-Cy5 and PTX3-PE were considered (light gray square). (B) Quantification of PTX3 binding among B cells before and after blocking of PTX3. Cy5, cyanin 5; PE, phycoerythrin. ^** < 0.01.

Figure 2

PTX3⁺ B cells are decreased in patients with lupus nephritis and are mainly confined to CD27⁻IgD⁺ B cells. (A) Absolute numbers of PTX3⁺ B cells (cell/mL) within (left) total; (middle) naïve or (right) memory B cells in HD (n = 22) and SLE (n = 26) and LN (n = 12) patients. (B) Frequencies of PTX3⁺ B cells (left), naïve (middle), and memory (right) are decreased in LN (n = 12) in comparison with HD (n = 22) and SLE (n = 26). (C) Distribution of CD27 and IgD expression by PTX3⁺ B cell subsets are shown. Enrichment in the naïve pool with decreases in the other subsets was found in HD (n = 22) and SLE (n = 26), but not in LN (n = 12). (D) Pie charts of percentages of PTX3⁺ CD27IgD subsets within the PTX3⁺ B cell pool are consistent with distribution of absolute numbers. Mann-Whitney U-test (^* < 0.05, ^** < 0.01, ^*** < 0.001). SLE, systemic lupus erythematosus; HD, healthy donors; LN, lupus nephritis; PTX3 pentraxin3.