AB0031 Deep immune-profiling of cd4+ t cells in behÇet’s disease
Authors: J. Nowatzky, Y. Xia, O. Manches
Journal: Annals of the Rheumatic Diseases (2018)

<h3>Background</h3> Functionality and immune-phenotypes of the human CD4<sup>+</sup> T-cell compartment in Behçet's disease (BD) are under-investigated, but several lines of evidence point to its relevance in the pathogenesis, progression and remission of the disease. <h3>Objectives</h3> We aimed to apply an unbiased single cell approach to assess protein expression levels of phenotypic and functional markers within the human CD4<sup>+</sup> T cell compartment in subjects with prototypical BD in order to identify cell populations of potential biologic relevance. <h3>Methods</h3> We determined single cell surface/intra nuclear expression levels of CD3, CD4, CD8, CD127, CD25, CD45RA, CCR7, FoxP3, HELIOS, Ki67, HLA-DR, CD38, CD39 in PBMC by flow cytometry and computed the representation of all mathematically possible cell populations within pre-defined starting populations. PBMC from BD subjects (n=13), healthy donors (HD) (n=25) and diseased subjects with non-BD auto-immune uveitis (n=11; VKH, Sarcoidosis, and HLA-B27 associated uveitis) were used. BD subjects met ISG criteria and were Arab or Chinese. 62% had pan-uveitis, 23% major vascular disease, and 7% parenchymal CNS disease. 46% were HLA-B51 carriers. <h3>Results</h3> Computation of all populations defined by CD127, CD25, CD45RA, CCR7, FoxP3, HELIOS, Ki67, HLA-DR within the CD4<sup>+</sup> T cell compartment yielded a total of 6560 (3^8–1) cell populations out of which 45 reached a significance level of p≤0.000001 in ANOVA testing to differentiate 3 groups (BD, non-BD uveitis, and HD). All of these populations comprised sub-types of the human regulatory T (Treg) cell compartment with a strong predominance of non-proliferating, non-activated, FoxP3<sup>+</sup>HELIOS<sup>+</sup> Treg carrying central-memory phenotypes (CD45RA<sup>-</sup>, CCR7<sup>+</sup>). Unpaired testing of BD vs non-BD revealed 43 distinct cell populations at a significance level of p≤0.002 representing CD25<sup>+</sup> non-Treg; comparison of BD vs HD uncovered 58 populations at a significance level of p≤0.0001 representing FoxP3<sup>+</sup>HELIOS<sup>+</sup> subpopulations, and non-BD vs HD identified 61 populations at p≤0.001, comprising CD25<sup>+</sup>CD127<sup>±</sup> FoxP3<sup>±</sup>, but consistently HELIOS<sup>-</sup> cells. A separate analysis using CD38, CD39, CD227, TIGIT, CD45RA, CCR7 within the CD3<sup>+</sup>CD4<sup>+</sup>CD8<sup>-</sup>CD127<sup>-</sup>CD25<sup>+</sup> compartment that contains most human Treg, showed 48 populations (p≤0.0001) in 3-way comparison pointing to high significance of TIGIT and CD226 staining Treg subpopulations, and 18 populations with differential expression of CD39<sup>+</sup> between BD and non-BD diseased subjects. TIGIT and CD226 co-expressing Treg (CD127<sup>-</sup>CD25<sup>+</sup>) subpopulations also reached significance (p≤0.02) in a longitudinal paired analysis of 7 BD subjects in active vs inactive disease states, as did 56 out of 6560 populations within total CD4<sup>+</sup> T cells, mostly representing non-Treg cells in active BD. <h3>Conclusions</h3> Differential expression of CD4<sup>+</sup> Treg and non-Treg cells shapes the immune-phenotype of BD vs states of health and non-BD autoimmune diseases that have phenotypic overlap with BD (uveitis). The populations with the highest significance for differentiating BD from healthy states seem to exist within the HELIOS<sup>+</sup>FOXP3<sup>+</sup> compartment of non-activated, non-proliferating Treg, suggesting relevance of a true Treg phenoptype. This is likely not specific to BD. Non-Treg CD25<sup>+</sup> cell populations seem more indicative of BD vs non-BD uveitic disease as well as of clinically active BD while populations with high CD39 expression may signify non-BD states of immune-mediated disease. <h3>Disclosure of Interest</h3> None declared