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An Adoptive Transfer Model of Rheumatoid Arthritis in Mice
Published on: June 6, 2025
Rheumatoid arthritis synovial fluid shows enrichment of T-cells producing GMCSF which are polyfunctional for TNFα and
Aastha Khullar1, Varun Dhir2, Biman Saikia3
1Division of Rheumatology, Department of Internal Medicine, PGIMER, Chandigarh, India.
Objectives:
GMCSF+T-cells may be involved in pathogenesis of rheumatoid arthritis (RA), and polyfunctionality may be a marker of pathogenicity. Although, higher frequencies of CD4+GMCSF+ T-cells have been reported, there are no data on CD8+GMCSF+ T-cells or polyfunctionality.Our objective was to enumerate frequencies of CD8+GMCSF+ T cells in RA blood and synovial fluid (SF), and assess their polyfunctionality, memory phenotype and cytotoxic ability.
Methods:
This study included RA patients (blood samples,in some with paired synovial fluid (SF)), healthy controls (HC) (blood) and SpA patients (SF). In some RA patients' blood was sampled twice, before and 16-24 weeks after methotrexate (MTX) treatment. After mononuclear cell isolation from blood and SF, ex-vivo stimulation using PMA/Ionomycin was done, and cells were stained (surface and intracellular after permeabilisation/fixation). Subsequently, frequencies of GMCSF+CD8+ and CD4+ T-cells, polyfunctionality (TNFα, IFNγ, IL-17), phenotype (memory) and perforin/granzyme expression were assessed by flowcytometry.
Results:
There was no significant difference in frequencies of GMCSF+CD8+ (3.7, 4.1%, p=0.540) or GMCSF+CD4+ T-cells (4.5, 5.2%, p=0.450) inblood of RA and HC. However, there was significant enrichment of both CD8+GMCSF+ (5.8, 3.9%, p=0.0045) and CD4+GMCSF+ (8.5, 4.5%, p=0.0008) T-cells inSF compared to blood in RA patients. Polyfunctional triple cytokine positive TNFα+IFNγ+GMCSF+CD8+T-cells (81, 36%, p=0.049) and CD4+T-cells (48, 32%, p=0.010) was also higher in SF compared to blood in RA. CD8+ T cells showed higher frequency of effector-memory phenotype and granzyme-B expression in RA-SF. On longitudinal follow-up, blood CD4+GMCSF+ T-cells significantly declined (4.6, 2.9%, p=0.0014) post-MTX.
Conclusions:
We report a novel finding of enrichment of CD8+GMCSF+ in addition to CD4+GMCSF+ T-cells in RA-SF. These cells showed higher polyfunctionality for TNFα and IFNγ, and effector memory phenotype suggesting their involvement in RA pathogenesis.
Insights
CD8+GMCSF+ T-cells are enriched in rheumatoid arthritis synovial fluid and exhibit polyfunctionality, suggesting a role in disease pathogenesis. Methotrexate treatment reduced CD4+GMCSF+ T-cells in blood.
Area of Science:
- Immunology
- Rheumatology
- Cell Biology
Background:
- Granulocyte-macrophage colony-stimulating factor (GMCSF)-producing T-cells are implicated in rheumatoid arthritis (RA) pathogenesis.
- While CD4+GMCSF+ T-cells are studied, CD8+GMCSF+ T-cells and their polyfunctionality in RA remain unexplored.
Purpose of the Study:
- To quantify CD8+GMCSF+ T-cells in RA blood and synovial fluid (SF).
- To assess the polyfunctionality, memory phenotype, and cytotoxic potential of these cells.
- To evaluate the impact of methotrexate (MTX) treatment on T-cell populations.
Main Methods:
- Flow cytometry was used to analyze T-cells from RA patients (blood and SF), healthy controls, and SpA patients.
- Cells were stimulated ex-vivo, stained for intracellular cytokines (TNFα, IFNγ, IL-17, GMCSF), and surface markers.
- Phenotypic analysis included memory markers and cytotoxic molecules (perforin, granzyme B).
Main Results:
- No significant difference in GMCSF+ T-cell frequencies in the blood of RA patients and healthy controls.
- Significant enrichment of both CD8+GMCSF+ and CD4+GMCSF+ T-cells was observed in RA synovial fluid compared to blood.
- Polyfunctional (TNFα+IFNγ+GMCSF+) CD8+ and CD4+ T-cells were higher in RA SF. CD8+ T-cells in RA SF showed increased effector-memory phenotype and granzyme-B expression.
- Methotrexate treatment led to a significant decrease in blood CD4+GMCSF+ T-cells.
Conclusions:
- A novel enrichment of CD8+GMCSF+ T-cells, alongside CD4+GMCSF+ T-cells, was found in RA synovial fluid.
- These enriched cells display heightened polyfunctionality and an effector-memory phenotype, implicating them in RA pathogenesis.
- The findings suggest a potential role for CD8+GMCSF+ T-cells in the inflammatory processes of rheumatoid arthritis.
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