CX3CR1+ age-associated CD4+ T cells contribute to synovial inflammation in late-onset rheumatoid arthritis

Abstract

Background: Recent evidence suggests that clonally expanded cytotoxic T cells play a role in various autoimmune diseases. Late-onset rheumatoid arthritis (LORA) exhibits unique characteristics compared to other RA forms, suggesting distinct immunological mechanisms. This study aimed to examine the involvement of cytotoxic T cells in LORA.

Methods: Fresh peripheral blood samples were collected from 78 treatment-naïve active RA patients, 12 with difficult-to-treat RA, and 16 healthy controls. Flow cytometry was employed to measure the proportions of CX3CR1⁺cytotoxic CD4⁺ and CD8⁺ T cells in these samples. Additionally, immunohistochemical staining was performed on lymphoid node and synovial biopsy samples from patients with RA.

Results: CX3CR1⁺cytotoxic CD4⁺ T cells were specifically increased in untreated, active patients with LORA, displaying features of CXCR3^mid age-associated T helper cells known as "ThA". CX3CR1⁺CD4⁺ T cells were identified as a cytotoxic ThA subset, as nearly all of these cells specifically expressed granzyme B. These cells were observed in enlarged lymph nodes and were found to infiltrate synovial tissues from patients with LORA. The proportions of CX3CR1⁺CD4⁺ T cells positively correlated with arthritis activity in LORA. The number of cells decreased after treatment with methotrexate, tumor necrosis factor inhibitors, and interleukin-6 inhibitors, whereas T-cell activation modulators did not affect them. Moreover, PD-1⁺CD38⁺CX3CR1⁺CD4⁺ T cells were identified as a treatment-resistant T cell subset that was characteristically increased in difficult-to-treat RA. CX3CR1⁺CD8⁺ T cells showed no significant difference between RA patients and healthy individuals, and no correlation with disease activity was observed. However, a correlation with age was observed in RA patients.

Conclusions: Our findings suggest that the immunopathogenesis of RA differs by age of onset, with CX3CR1⁺ age-associated cytotoxic CD4⁺ T cells playing a significant role in LORA. Additionally, the presence of a specific CX3CR1⁺ T cell subset may be linked to treatment resistance.

Keywords: Aging; CX3CR1; Cytotoxic T cells; Difficult-to-treat rheumatoid arthritis; Late-onset rheumatoid arthritis; Rheumatoid arthritis.

Fig. 1

Peripheral blood CX3CR1⁺ T cells are increased in LORA. a Gating strategy for identifying CX3CR1⁺ T cells by flow cytometry. b Comparison of peripheral blood CX3CR1⁺ T cells between treatment-naive, active RA, and HC. Mann–Whitney U test. c Comparative analysis of peripheral blood CX3CR1⁺ T cells among clinical characteristics of RA (LORA, seropositivity, and sex difference). Seropositivity for autoantibodies refers to the presence of either RF, anti-CCP antibodies, or both. Mann–Whitney U test. d Intracellular GZMB expression in CX3CR1⁺CD4⁺ T cells. The area within the black bold frame represents CX3CR1⁺GZMB⁺CD4⁺ T cells

Fig. 2

CX3CR1⁺ T cells exhibit characteristics of age-associated T cells. a Correlation analysis between the proportion of CX3CR1⁺ T cells and age in patients with RA (n = 78). Spearman’s correlation test. b Flow cytometry analysis of the expression of age-associated molecules (CD27, CD28, CD96, CD57) in CX3CR1⁺ T cells from peripheral blood of LORA patients (n = 7). Paired t-test. c Analysis of CD95 expression in CX3CR1⁺ T cells from peripheral blood of LORA

Fig. 3

CX3CR1⁺ T cells exhibit features of CXCR3^mid age-associated chemokine receptor expression. a Unbiased analysis using flow cytometry and tSNE of chemokine receptor expression (CCR7 for lymph node-related, CCR4 for Th2 or Treg-related, CCR6 for Th17-related, CXCR5 for Tfh-related) in CX3CR1⁺ T cells. b Analysis of PD-1 expression in CX3CR1⁺CD4⁺ T cells from patients with LORA (n = 3). c Analysis of chemokine receptor expression related to Th1 (CXCR3^high) or age-associated (CXCR3^mid). d Phenotypic analysis of CX3CR1⁺ CD4⁺ or CX3CR1⁺ CD8⁺ T cells (EM or TEMRA). Paired t-test

Fig. 4

Identification of CX3CR1⁺ T cells in enlarged lymph nodes and synovial inflammatory sites. a, b Comparative analysis of the proportion of patients with lymphadenopathy on CT imaging in LORA and non-LORA groups. Fisher’s exact test. c Distribution of enlarged lymph nodes. d Immunostaining of CX3CR1⁺ CD4⁺ T cells and CX3CR1⁺ CD8⁺ T cells in lymph nodes. e. Immunostaining of CX3CR1⁺ CD4⁺ T cells and CX3CR1⁺ CD8⁺ T cells in synovial inflammatory tissues from LORA patients. Scale bar shows 100 μm

Fig. 5

Association of disease activity with CX3CR1⁺ T cells and impact of treatment. a Heatmap of correlation analysis between peripheral blood CX3CR1⁺ CD4⁺ T cells, CX3CR1⁺ CD8⁺ T cells, and clinical indicators. Spearman's correlation analysis. b Longitudinal changes of CX3CR1⁺ T cells after initiation of treatment (n = 30). Wilcoxon's rank sum test. c Heatmap showing longitudinal changes of CX3CR1⁺ CD4⁺ T cells and CX3CR1⁺ CD8⁺ T cells analyzed based on specific treatments

Fig. 6

CX3CR1⁺ T cells remain elevated and activated in treatment-resistent RA. a Comparative analysis of peripheral blood CX3CR1⁺ CD4⁺ T cells and CX3CR1⁺ CD8⁺ T cells in D2T RA, non-D2T RA, and HC. One-way ANOVA followed by Tukey's multiple comparison test. b Heatmap showing correlations between clinical indicators and CX3CR1⁺ CD4⁺ T cells, CX3CR1⁺ CD8⁺ T cells in D2T RA patients. Spearman's correlation analysis. c Comparative analysis of activation markers (PD-1, CD38, CD25, HLA-DR) and phosphorylated STAT1 in CX3CR1⁺ T cells between D2T RA (n = 6) and non-D2T RA (n = 6) patients. t test