PD-1 Marks Senescent T Cells That Drive Rheumatoid Arthritis via Mitochondrial Breakdown

Rheumatoid arthritis (RA) is an autoimmune disease driven in part by dysregulated CD4+ T cells that acquire premature senescence features. While senescent T cells are known to promote inflammation through their senescence-associated secretory phenotype (SASP), the precise subset responsible and the underlying mechanism have remained unclear. This study from Dalian Medical University identifies CD4+PD-1+ T cells as the critical pathogenic senescent subset and maps out a mechanistic axis — PD-1 → DRP1 suppression → impaired mitophagy → mitochondrial ROS accumulation → SASP — that drives RA progression.

The researchers enrolled RA patients meeting ACR 1987 criteria and age- and sex-matched healthy controls. Flow cytometry confirmed a significant expansion of CD4+PD-1+ T cells in RA peripheral blood. These cells displayed classical senescence hallmarks compared with CD4+PD-1− T cells from the same patients: elevated SA-β-galactosidase activity, reduced Ki67 proliferation marker, increased expression of p16, p21, and p53, loss of CD28 co-stimulatory molecule, and upregulation of SASP cytokines including IL-1β, IL-6, IL-8, TNF-α, and MMP-3. The PD-1+ subset also showed impaired proliferative capacity by CFSE dilution assay and elevated cytotoxic markers (perforin, granzyme B), painting a picture of cells that have stopped dividing but remain metabolically destructive.

Mitochondrial phenotyping revealed that RA CD4+PD-1+ T cells harbored dysfunctional mitochondria: reduced mitochondrial membrane potential by JC-10 assay, altered morphology (rounder, less elongated aspect ratio by Mitotracker Green/ImageJ), and substantially elevated mitochondrial ROS (MtROS) measured by MitoSOX Red flow cytometry. Critically, these cells showed significantly lower DRP1 protein and mRNA expression and impaired mitophagy flux, quantified using the mt-mKeima adenoviral reporter system — a ratiometric pH-sensitive probe that distinguishes mitochondria in acidic lysosomes (active mitophagy, 550 nm excitation) from healthy ones (440 nm excitation). PINK1 and Parkin, the canonical mitophagy mediators, were also reduced.

To establish causality, the team performed siRNA knockdown of DRP1 and Parkin in Jurkat T cells, which recapitulated the MtROS accumulation and SASP induction seen in primary RA cells. Conversely, DRP1 overexpression (pcDNA3.1-DRP1) or treatment with the mitochondria-targeted antioxidant MitoQ (200 nM) significantly reduced MtROS and SASP markers. The DRP1 inhibitor mdivi-1 (1 μM), paradoxically, also reduced SASP in some contexts, suggesting complex dynamics. PD-1 ligation experiments using plate-bound PD-L1 or PD-L2 demonstrated that PD-1 signaling directly suppressed DRP1 transcription via inhibition of HIF-1α, a known transcriptional activator of DRP1.

In collagen-induced arthritis (CIA) mouse models, adoptive transfer of 1×10⁶ CD4+PD-1+ T cells from CIA spleens significantly accelerated disease progression compared with CD4+PD-1− T cell transfer, as measured by paw thickness, clinical arthritis scores, and histological synovitis/cartilage damage (H&E and safranin O staining). Pre-treatment of CD4+PD-1+ T cells with MitoQ before transfer attenuated the disease-accelerating effect, validating MtROS as a functional driver. Coculture experiments showed that CD4+PD-1+ T cells drove greater plasmablast differentiation from B cells, induced higher IL-1β and IL-6 expression in RA fibroblast-like synoviocytes, and caused greater chondrocyte apoptosis than CD4+PD-1− T cells — collectively explaining their tissue-destructive capacity. These findings establish a therapeutically actionable PD-1–DRP1–mitophagy–SASP axis in RA.