A5.26 Regulation of Expression and Function of Negative Immunomodulatory Receptors in B-Cells: Implications for the Pathogenesis of Systemic Lupus Erythematosus
Authors: Amalia Zampoylaki, Christianna Choulaki, Irini Gergianaki, Garyfalia Papadaki, Antonis Fanouriakis, Prodromos Sidiropoulos, Dimitrios T Boumpas, George Bertsias
Journal: Annals of the Rheumatic Diseases (2013)

<h3>Background and Objectives</h3> Fine tuning of B-cell activation and differentiation depends on convergent signals from the B-cell receptor (BCR), costimulatory/coinhibitory membrane receptors, and Toll-like receptors. We sought to examine the expression and function of the coinhibitory receptors programmed death-1 (PD-1), PD-1 ligand-1 (PD-L1), B and T lymphocyte attenuator (BTLA) in B-cells from healthy donors, and from patients with systemic lupus erythematosus (SLE), the prototype of systemic autoimmune disease characterised by activated B-cells and production of high-titer autoantibodies by long-lived plasma cells. <h3>Materials and Methods</h3> Peripheral blood CD19<sup>+</sup> B cells were purified from healthy donors (n = 11) and active SLE patients (n = 15; SLE disease activity index 8.3 ± 2.7 [mean ± SEM]). PD-1, PD-L1, and BTLA were examined by flow cytometry in naïve (CD19<sup>+</sup>CD27<sup>–</sup>), memory/transitional (CD19<sup>+</sup>CD27<sup>+</sup>), and plasma B-cells (CD19<sup>+</sup>CD27<sup>hi</sup>) at baseline and following stimulation. Activation, differentiation, and proliferation (CFSE dilution) of B-cells were examined in the presence or absence of the BTLA ligand, HVEM. Western blot was used to assess the phosphorylation of intracellular kinases. <h3>Results</h3> In healthy donors, the coinhibitory receptors PD-1 and PD-L1 were significantly upregulated on circulating plasma-cells compared to transitional/memory and naïve B-cells (PD-1: 36 ± 7%, 14 ± 3%, 2.0 ± 0.5%; PD-L1: 94 ± 2%, 83 ± 5%, 62 ± 8%, respectively, p &lt; 0.001). BTLA was expressed by 93–100% of B-cells, and mean fluorescence intensity was significantly higher in plasma-cells (334 ± 146 versus 127 ± 12 in naïve B-cells, p = 0.048). BCR activation enhanced the expression all three receptors in normal B-cells; addition of CpG-ODN (TLR-9 ligand) further induced PD-1 and PD-L1-but not BTLA–expression, whereas addition of the cytokines IL-4, IL-10, or IL-21 reduced PD-1 and BTLA levels. In vitro crosslinking of BTLA resulted in reduction of BCR-induced phosphorylation of ERK, CD80/CD86 and BAFF-receptor expression, as well as in inhibition of cell proliferation (divided cells: 5.3 ± 0.4% versus 17.7 ± 0.1% in anti-IgM-stimulated cells). In comparative analysis, SLE patients exhibited significantly higher PD-1 expression on plasma-cells compared to healthy donors (65 ± 5% versus 36 ± 7%, p = 0.002), whereas there was no difference in PD-L1 or BTLA. Preliminary studies suggest distinct roles for PD-1 and BTLA in regulation of activation and maturation of B-cells in healthy controls and in the context of lupus. <h3>Conclusions</h3> The coinhibitory receptors PD-1, PD-L1 and BTLA demonstrate differential expression among B-cell subsets and they are induced upon stimulation with important implications for the regulation of B-cell activation, proliferation and differentiation. Aberrancies in the expression and function of coinhibitory receptors in SLE plasma B-cells could contribute to enhanced autoantibody-forming capacity and disease pathogenesis.