NET-ting the autoreactive B cell memory by therapeutically targeting the humoral autoimmunity in patients with SLE

This consortium aimed at investigating the humoral autoimmune response in three different SLE patient cohorts treated with specific B cell-targeted therapies, i.e. Rituximab, Bortezomib and their combination.

The humoral autoimmune response was studied on different aspects in SLE patients before and after therapy, as follows:

• The induction of neutrophil extracellular traps to quantify the autoantigenic load of nuclear material;
• Degradation of neutrophil extracellular traps by SLE sera to quantify the autoantigenic load of nuclear material;
• Autoantibodies recognizing dsDNA, nucleosomes, histones, alphaenolase and C1q to quantify the humoral autoimmune products;
• Autoantigen-specific B cells recognizing dsDNA, nucleosomes, histones, alphaenolase and C1q to quantify the humoral autoimmune memory.

The project validated novel assays for autoantigen monitoring in SLE patients in relation to treatment and clinical response. As such the researchers were able to combine autoantigen monitoring with autoantibody monitoring in SLE patients that were treated with RTX, RTX+BLM and BTZ, novel B-cell targeted strategies that differentially target B cell and plasma cell subsets. In a reverse translational study, it was demonstrated that autoantibody levels decreased upon each treatment strategy, but the extent of targeted autoantibodies was most significant for RTX+BLM in a quantitative manner (reduced autoantibody repertoire) as well as a qualitative manner (reduced low, medium and highavidity anti-dsDNA autoantibodies). These effects were less pronounced for RTX only and not observed in BTZ-treated patients. Especially the reversal of anti-C1q to seronegative was associated with reduced IC-mediated inflammation and clinical disease activity, which happened most frequent after RTX+BLM, less after RTX and not after BTZ treatment. Lastly, hints of persisting
autoreactive memory in SLE patients were found despite a clinical response to B-cell targeted therapy. These observations collectively demonstrated the relevance of in-depth monitoring of the immunological effects of B-cell targeted strategies that have potential implications for the clinic.

Patients with SLE typically have circulating autoantibodies against DNA as a result of a humoral (auto-)immune response. This research project has performed a comprehensive, reverse translational study to better understand the pathophysiology of the humoral autoimmune response in SLE patients. As such this project has monitored SLE-relevant autoantibodies as well as autoantigens in 42 refractory SLE patients with renal involvement who were treated with experimental treatment regimens (i.e. rituximab, bortezomib or combination rituximab + belimumab). We found that although each treatment strategy reduced autoantibody levels there were significant differences between these treatments and between patients. In general, achieving a reduction of autoantibody load, and ultimately achieving negativity of autoantibodies, and autoantigenic load was associated with beneficial clinical outcome and could be a key treatment target in SLE patients.
Altogether this project has established new ways to monitor autoantigens, autoantibodies and autoantibody-producing cells in SLE patients within the context of B-cell-targeted treatment strategies. As such, we have found hints of minimally residual autoimmunity after treatment despite clinical response to that treatment. Future studies should be aimed at applying these novel immunomonitoring tools to better detect en investigate MRA in SLE patients.

• van Dam LS, Osmani Z, Kamerling SWA, et al. A reverse translational study on the effect of rituximab, rituximab plus belimumab, or bortezomib on the humoral autoimmune response in SLE, Rheumatology, Volume 59, Issue 10, October 2020, Pages 2734–2745, doi:10.1093/rheumatology/kez623
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• Dam, Kraaij, T., Kamerling, S. W. A., Bakker, J. A., Scherer, U. H., Rabelink, T. J., Kooten, C., & Teng, Y. K. O. (2019). Intrinsically Distinct Role of Neutrophil Extracellular Trap Formation in Antineutrophil Cytoplasmic Antibody–Associated Vasculitis Compared to Systemic Lupus Erythematosus. Arthritis & Rheumatology (Hoboken, N.J.), 71(12), 2047–2058. https://doi.org/10.1002/art.41047 
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• Arends, E. J., van Dam, L. S., Kraaij, T., Kamerling, S. W. A., Rabelink, T. J., van Kooten, C., Teng, Y. K. O. A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation. J. Vis. Exp. (143), e59150, doi:10.3791/59150 (2019). 
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• Van Dam, L. S., Rabelink, T. J., van Kooten, C., & Teng, Y. (2018). Clinical Implications of Excessive Neutrophil Extracellular Trap Formation in Renal Autoimmune Diseases. Kidney international reports, 4(2), 196–211. doi: 10.1016/j.ekir.2018.11.005.
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EULAR Abstract

• Van Dam L, Osmani Z, Kraaij T, et al. FRI0311 The effect of b cell targeted therapies on autoantibodies and excessive neutrophil extracellular trap formation in systemic lupus erythematosus patients. Annals of the Rheumatic Diseases 2018;77:692.
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• Dam LV, Kraaij T, Kamerling S, et al. SAT0015 Anca-associated vasculitis- and systemic lupus erythematosus-induced neutrophil extracellular traps have intrinsically different features. Annals of the Rheumatic Diseases 2017;76:774.
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The experimental nature of our research proposal limits the potential contribution of patient research partners. However, patient representatives were involved in the separate clinical trials at each collaborating centre which investigate therapeutic strategies that specifically target humoral autoimmunity. In addition, the project results are communicated to lupus patient organisations through lay summaries in patient magazines and presentations at meetings.