Background/Purpose:

We have previously identified two dichotomous CD4 pathogenic subsets in both T effector (CPLs: Circulating pathogenic like lymphocytes) and T regulatory (iaTreg: inflammation associated Treg) compartments that are both HLA-DR⁺, antigen experienced, pro-inflammatory, correlating with disease activity and sharing strong TCR sequence oligoclonality with synovial T cells from JIA patients. Despite being two functionally disparate T cell subsets, their phenotype and association with clinical fate suggests that these functionally discordant subsets may originate from a common precursor. To elucidate the common pathogenic gene drivers and their associated network of pathways within these two pathogenic subsets, we decided to perform next generation RNA sequencing on sorted CPLs and iaTregs and their conventional Teff/Treg counterparts.

Methods:

CPLs were sorted as CD3⁺ CD4⁺ CD14^– HLADR⁺ CD25/CD127 Teff gate, and iaTregs were sorted as CD3⁺ CD4⁺ CD14^– HLADR⁺ CD25^hi/CD127^low Treg gate with FACs Aria II from n=8 active JIA PBMCs. As a comparative control, similar HLADR^– counterparts were respectively sorted from the same patients. Sorted cells were lysed and extracted for RNA, and cDNA libraries were ran on the Illumina HiSeq High output platform. RNA-Seq raw reads were mapped to human genome using STAR aligner with default options and reads were summarised at gene level by feature Count programme. Differential expression analyses were performed using edgeR package, and pathway enrichments under R statistical environment and Reactome.

Results:

Comparative gene expression analysis, between CPLs vs iaTreg and that of the common pool of Teff vs Treg, and phylogenetic association analysis suggests both CPLs and iaTreg are uncoupled and group separately from their conventional T cell compartments. TCR sequence oligoclonality in CPLs/iaTregs versus that of the common Teff/Treg pool reinforce the presence of a common selection pressure. Pathway enrichment analysis reveal similar dysregulated pathways (IFN-g, PD1, CD28 costimulation) within T cell signalling. Furthermore, weighted gene correlation network analysis (WGCNA) identified strongly coordinated HLA-DR gene network modules and suggests these networks as potential drivers of pathogenic T cell subsets from conventional subsets. Gene set enrichment analysis suggest that HLA-DR module genes are involved in TNFA signalling, inflammatory response, complement, and apoptosis. Transcription factors (TFs) gene regulatory network (TF-GRN) analysis identified the key regulatory molecules driving the convergence of pathogenic CPL and iaTreg populations. Global TF-GRN network analysis identified FOXP3, CEBP, SPI and E2F1 as key TFs driving the Teff, Treg to pathogenic iaTreg and CPLs, respectively.

Conclusion:

These mechanistic data strongly suggest shared pathogenic and developmental pathways for two T cell subsets and are strongly associated with clinical fate in autoimmune human arthritis. These pathways are probably shaped from a shared precursor in an inflammatory microenvironment.