Characterisation of Programmed Cell Death Protein 1 Expressing Peripheral T Helper Cells in the Early Rheumatoid Arthritis Synovium.

Aim: Programmed cell death protein 1 (PD-1) expressing T cells are associated with the pathogenesis of autoimmune inflammatory diseases such as rheumatoid arthritis (RA). Recently, a new subset of CXCR5- T cells, termed T peripheral helper (Tph) cells, distinct from CXCR5+ T follicular helper (Tfh) cells, have been identified in established RA synovial tissue (ST). However, it remains unknown whether Tph cells accumulate in treatment-naïve early RA prior to fully established disease. We aimed to identify Tph cells in treatment-naïve early RA patients using flow cytometry (FCM) and multi-parameter immunofluorescence (IF) microscopy from ST and matched peripheral blood mononuclear cells (PBMCs).

Methods: FCM: Dissociated ST (n = 4), and PBMCs (n = 7) were stained with Zombie UV® viability dye, prior to immunostaining with anti-CD45RO, PD1, CD3, ICOS, CD8, CD4, CD20, CXCR5, TIGIT and CD38 antibodies, and analysed using FlowJo 10.5.2. Histology: 4𝜇m thick RA ST sections were prepared for Opal™ multispectral IF. Primary antibodies for CD45RO, CD20, PD-1 and CXCR5 were sequentially stained, each followed by HRP amplification and specific Opal™ reactive fluorophores. Nuclei were stained with DAPI prior to acquisition on the Vectra 3.0 imaging system. Images were processed and analyzed using InForm®.

Results: We observed a higher frequency of PD1hi CXCR5- Tph in RA ST and PBMCs versus controls, while no significant difference in Tfh frequency was observed. Furthermore, IF identified a 10-fold increase of Tph cells in early RA ST follicular and diffuse regions and identified Tph in proximity to GC B cells.

Conclusions: Here, we demonstrate for the first time, the presence and potential role of Tph cells in early RA, with Tph accumulation and close association with B cells, potentially contributing to disease pathogenesis and progression. Tph cells may be a novel immune cell target to therapeutic interception of disease progression and warrant further study in early RA.