1565: Dissection of Ocular and Peripheral Blood Immune Phenotypes Suggests Preferential Transmigration of CD16⁺ Monocytes to Effector Sites in Behçet’s Disease

Yesim Ozguler¹, Ziyan Lin², Ann Cavers³, gulen Hatemi⁴, Olivier Manches⁵ and Johannes Nowatzky³, ¹Istanbul University-Cerrahpasa, Cerrahpasa Medical Faculty, Department of Internal Medicine, Division of Rheumatology, New York, NY, ²NYU Grossman School of Medicine, NYU Langone Applied Informatics Laboratories, New York, NY, ³NYU Grossman School of Medicine, New York, NY, ⁴Istanbul University-Cerrahpaşa, Cerrahpasa Medical Faculty, Department of Internal Medicine, Division of Rheumatology, Istanbul, Turkey, ⁵Recherche et Développement, Immunobiology and Immunotherapy in Chronic Diseases, Institute for Advanced Biosciences, Inserm U 1209, CNRS UMR 5309, Université Grenoble Alpes Etablissement Français du Sang Auvergne-Rhône-Alpes, Grenoble, France

Background/Purpose: Cellular immunity of Behçet's Disease (BD) remains poorly understood. Previous work has provided clues pointing to most innate and adaptive immune cell types in BD, but strong signals from non-immunogenetic studies are rare and often inconclusive. Here we aimed to identify highly BD/ healthy donor (HD) discriminant immune cells in semi-biased and targeted approaches and determine their significance at a BD-relevant effector site: the eye.

Methods: We utilized multi-parametric flow cytometry to dissect cellular phenotypes in peripheral blood mononuclear cells (PBMC) of untreated BD patients (n=27) and HD (n=22) consisting predominantly of active ocular and major vascular BD subjects. Data were subjected to supervised machine learning (CITRUS), and results were verified with targeted gating. We also assessed anterior chamber (AC) fluid cells and autologous PBMC from BD uveitis patients with single-cell RNA-sequencing.

Results: CITRUS identified CD16+, CD14low, CD4low, CD3-, CD19- cells as the only BD/HD discriminant cellular expression pattern at an FDR of < 0.05. Targeted gating confirmed highly significant differences with large effect sizes in PBMC of BD vs. HD for "non-classical" (CD14lowCD16hi, p< 0.0001) and "intermediate" (CD14+CD16+, p=0.0006) monocytes at decreased frequencies for BD in peripheral blood. CD16+ dendritic cells (DC) were also significantly decreased in BD PBMC vs. HD. The effect sizes and significance levels in the CD16+ DC comparisons were inversely proportional to the level of HLA-DR expression. "Classical" (CD14++CD16-) monocytes were significantly more frequent in BD PBMC than in HD (p=0.021), but with smaller effect sizes. CD14+ cells showed a strong presence in the AC of the eye during BD uveitis and co-expressed CD16 far more frequently than CD14+ cells in autologous peripheral blood.

Conclusion: Significant differences in frequencies of CD16+ monocyte and DC subsets with large effect sizes in the peripheral blood of untreated active BD patients vs. HD point to their importance for the patho-immunobiology of BD. The high abundance of CD14+ cells with CD16 co-expression in the AC of the eye during uveitis, contrasted with their low relative frequencies in autologous peripheral blood, strongly suggests their transmigration into the eye during BD uveitis rather than a stochastic process.

Disclosures: Y. Ozguler, UCB, Novartis, Pfizer; Z. Lin, None; A. Cavers, None; g. Hatemi, Celgene, UCB, Novartis, AbbVie/Abbott; O. Manches, None; J. Nowatzky, None.