Background/Purpose: Functionality and immune-phenotypes of the human CD4⁺ T-cell compartment in Behçet’s disease (BD) are under-investigated, but several lines of evidence point to its relevance in the pathogenesis and progression of the disease. We aimed to apply an unbiased single cell approach to dissect the immune-phenotype of CD4⁺ T cells in prototypical BD in order to identify cell populations of potential pathobiological relevance.

Methods: We determined single cell expression levels of CD3, CD4, CD8, CD127, CD25, CD45RA, CCR7, FoxP3, HELIOS, Ki76, HLA-DR, CD38, CD39 in PBMC by flow cytometry and computed the representation of all possible cell populations within CD4⁺ starting populations in PBMC from healthy (HD, n=25), BD (n=13), and diseased subjects with non-BD auto-immune uveitis (n=11, VKH, Sarcoidosis, and HLA-B27 associated uveitis). 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.

Results: Computation of all populations defined by 8 markers (CD127, CD25, CD45RA, CCR7, FoxP3, HELIOS, Ki67, HLA-DR) within the CD4⁺ T cell compartment yielded a total of 6,560 cell populations per subject out of which 45 reached significance (p<=0.000001) differentiating 3 groups (BD, non-BD uveitis, and HD). All of these populations comprised sub-types of the human regulatory T (Treg) cell compartment with strong predominance of non-proliferating, non-activated, FoxP3⁺Helios⁺ Treg carrying central-memory phenotypes (CD45RA^–, CCR7⁺). 2-group testing of BD vs non-BD revealed 43 distinct cell populations at a significance level of p <=0.002 representing CD25⁺ non-Treg; comparison of BD vs HD uncovered 58 populations at significance level of p <=0.0001 representing FoxP3⁺Helios⁺ subpopulations, and non-BD vs HD identified 61 populations at p<=0.001, comprising CD25⁺CD127^+/- FoxP3^+/-, but consistently HELIOS^–, presumably non-Treg populations. A separate analysis using 6 marker combinations (CD38, CD39, CD226, TIGIT, CD45RA, CCR7) within the CD3⁺CD4⁺CD8^–CD127^–CD25⁺compartment which contains most human Treg, showed 48 populations (p <=0.0001) in 3-way comparison (BD, non-BD uveitis, and HD) pointing to high significance of TIGIT and CD226, and 18 populations with differential expression of CD39⁺ between BD and non-BD diseases subjects. TIGIT and CD226 co-expressing Treg (CD127^–CD25⁺) subpopulations also reached significance (p<=0.02) in a longitudinal analysis of 7 BD subjects in active vs inactive disease states, as did 56 out of 6,560 populations within total CD4⁺CD3⁺ cells, mostly representing non-Treg cells in active disease.

Conclusion: Differential expression of CD4⁺ Treg and non-Treg cells shapes the immune-phenotype of BD vs HD and non-BD autoimmune diseases that have phenotypic overlap with BD (uveitis). Populations within the HELIOS⁺FOXP3⁺compartment of non-activated, non-proliferating Treg had the highest significance when differentiating BD from HD, suggesting relevance of a true Treg phenotype. Non-Treg CD25⁺ 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 indicate non-BD states.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/deep-immune-profiling-of-cd4-t-cells-in-behcets-disease/