Background/Purpose: CD4+ T cells play a key role in the initiation and progression of RA. Past studies have identified impaired function and regulation of several CD4+ T cell lineages in RA patients. However, how diverse cellular subsets are maintained has not been comprehensively analyzed. Here, we utilized mass cytometry to simultaneously assay expression of 36 markers to examine high-dimensional complexity of CD4+ T cells in RA patients.

Methods: Cryopreserved cells from 10 RA patients and 7 healthy controls were thawed, washed, and stimulated with 5ng/ml of PMA and 50ng/ml ionomycin in the presence of 2mM Monensin and 5ug/ml Brefeldin A at 37⁰C for 5 hours. After stimulation, cells were washed and incubated in 1uM cisplatin for 5 min, followed by staining with surface antibody cocktail for 30 min at room temperature. A panel of 36 metal conjugated antibodies was generated using the X8 Maxpar kit. For intracellular staining, cells were permeabilized and fixed using Foxp3 staining buffer set and incubated with the intracellular antibody cocktail for 1 hr at room temperature. After staining, cells were washed then resuspended in 2% paraformaldehyde with 125nM iridium intercalator for an overnight incubation at 4⁰C. The next day, cells were washed and resuspended in PBS containing normalization beads before acquisition on CyTOF 2.

Results: Our data show a prominent expansion of CD57⁺ CD4⁺ T cells in RA patients. CD57⁺ T cells express cytotoxic granules, perforin, granzymes A and B, and have been previously associated with more active and erosive disease in RA. Interestingly, we identified three distinct subsets of CD57⁺ CD4⁺ T cells and only those that co-express high levels of IFN-g and TNF-a are increased in RA patients. In addition, patient derived samples showed a decrease in several populations of cytokine negative CD45RA⁺ naïve T cells.

Conclusion: Pilot analysis on a small numbers of samples from RA patients and healthy controls revealed cellular complexity in CD4⁺ T cells and suggested unique changes in RA patients. Extension of this analysis to a large cohort of RA patients with well annotated disease characteristics may uncover novel biomarkers for detection of early disease and provide new insights into the pathogenesis of RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/mass-cytometry-analysis-of-cd4-t-cells-in-patients-with-rheumatoid-arthritis/