Background/Purpose:

We have previously identified two CD4 pathogenic circulatory subsets in both T effector (CPLs) and T regulatory (iaTreg) 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 distinct 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 in both the circulation and the synovial micro-environment.

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=16 active JIA PBMCs, n=8 paired JIA SFMCs, and n=8 healthy paediatric 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 conversion/amplification were then carried out using SMART-seq v4. Libraries are prepared and multiplexed using Nextera XT DNA library preparation kit, and ran on the Illumina HiSeq High output platform.

Results:

Comparative differential gene expression (DEG) reveal strong transcriptomic convergence between CPLs and iaTregs as compared with the common pool of Teff and Treg. Phylogenetic analysis indicate the convergence has uncoupled the CPLs or the iaTregs away from their respective original compartments (Teff or Treg) into a common branch point. Restriction in TCR sequence oligoclonality in CPLs/iaTregs versus that of the common Teff/Treg pool reinforce the possibility of a common selection pressure. Pathway enrichment analysis reveal similar dysregulated pathways (IFN-g, PD1, CD28 costimulation) within T cell signalling for both CPLs and iaTregs. Furthermore, Weighted gene correlation network analysis (WGCNA) identified strongly coordinated HLA-DR gene network module and suggests its potential role as the driver of pathogenic T cell subsets away from conventional subsets. Gene set enrichment analysis (GESA) suggest that HLA-DR module genes are involved in TNFA signalling, inflammatory response, complement, and apoptosis. Global transcription factors gene regulatory network (TF-GRN) analysis identified several key regulatory molecules (FOXP3, CEBP, SPI and E2F1) driving the convergence of pathogenic CPLs and iaTreg populations. Taken together , we have shown that reveal several layers of mechanism operate to drive the convergence of CPLs and iaTregs, suggesting the possibility of common disease drivers in active JIA patients.

Conclusion:

Overall the transcriptomic data indicate strong similarity in both pathogenic populations and underscore a potential mechanistic role of the inflammatory microenvironment in shaping two functionally dichotomic populations.

« Back to 2018 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/microenvironment-driven-re-shaping-of-pathogenic-t-effector-and-regulatory-subset-in-active-juvenile-idiopathic-arthritic-patients/