Expanded Peripheral T Helper Cells Characterize the Early Rheumatoid Arthritis Synovium.

Background/Purpose: Programmed cell death protein 1 (PD-1) expressing T-cells are implicated in the pathogenesis of autoimmune inflammatory diseases such as rheumatoid arthritis (RA). In secondary lymphoid organs, CXCR5+ T follicular helper (TFH) cells secrete IL-21 and provide co-stimulatory signals to germinal centre (GC) B cells, allowing for affinity maturation of antibody responses. Recently a new subset of CXCR5- T-cells, termed T peripheral helper (TPH) cells driving B cell differentiation, have been identified in ectopic lymphoid structures in established RA synovial tissue (ST). Critically, 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 (FACS) and multi-parameter immunofluorescence (IF) microscopy from ST and matched peripheral blood mononuclear cells (PBMCs).

Methods: FACS: Fresh dissociated ST (n=4), and PBMCs (n=7) single cell suspensions were stained with Zombie UV®, CD45RO, PD1, CD3, ICOS, CD8, CD4, CD20, CXCR5, TIGIT and CD38 and analysed using FlowJo 10.5.2.

Histology: 4????m 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. DAPI nuclear staining was performed prior to image acquisition on the Perkin Elmer Vectra 3.0 imaging system. Images were processed and analyzed using InForm®, a machine learning software package, where the tissue segmentation and TPH and TFH cells quantification was performed.

Results: ST FACS demonstrated T cell infiltration in ST with differential expression of PD-1, CD45RO, ICOS, TIGIT and CD38 (Figure 1A). We observed a higher frequency of PD1hi CXCR5- TPH in RA ST and PBMCs vs. controls (Figure 1B-D). Importantly, no significant difference in TFH frequency was observed in RA and controls. Furthermore, IF identified a 10-fold increase of TPH cells in early RA ST follicular and diffuse regions and identified TPH adjacent to GC B cells (Figure 1E-I).

Conclusion: In this study, we demonstrate, for the first time, the presence and potential role of TPH cells in early RA, with TPH accumulation and close interaction with GC 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.