Background/Purpose

Immunosenescence accompanied by accumulation of senescent T cells  is a hallmark feature in the pathogenesis of rheumatoid arthritis (RA). Here we characterize a novel senescent regulatory T cell (Tregs, CD4⁺CD28^–FoxP3⁺) subset in RA patients.

Methods

Prospective, cross-sectional study on 35 patients with RA [mean age 58 (±SD 9.5), 71.4% female, SDAI 8.15 (±1.2)] and 25 healthy controls [HC, mean age 56.4 (±6.7), 60% female]. We used flow cytometry to determine the prevalence of senescent CD4⁺CD28^–FoxP3⁺ T cells and to characterize their phenotype, proliferation, cytokine production and apoptosis. T cell receptor diversity was determined by RT-PCR. In vitro generation of senescent Tregs was performed in cell culture experiments using magnetic bead isolated CD4⁺CD25⁺CD127^lowTregs and stimulation with anti-CD3/CD28 beads, interleukin (IL) -2 with or without TNF-α (100ng/ml) for 14 days.

Results

Two percent [±2.8] of CD4⁺ T cells were CD28^–FoxP3⁺ in RA patients whereas this subset was almost absent in HC [0.6 (±0.8), p=0.077]. The number of CD4⁺CD28⁺FoxP3⁺ Tregs was comparable in both groups [28.6 (±18.5) vs. 32.7 (±18), p=0.480]. In vitro assays showed that exposure of CD4⁺CD28⁺ Tregs to TNF-α led to a downregulation of CD28 and thus to the CD28^–FoxP3⁺ phenotype.

Surface receptor expression analysis of CD28^–FoxP3⁺ and CD28⁺FoxP3⁺ Tregs demonstrated that CD28^–FoxP3⁺ cells expressed higher levels of the regulatory protein PD-1 [17.45% (0-36.4) vs. 5.45% (1.8-13.5), p=0.034], whereas CTLA-4 expression was similar in both subsets. Production of various cytokines including IL-2, IL-4, IL-10, IL-17, TNF-α and IFN-γ was increased in CD28^–FoxP3⁺ compared to CD28⁺FoxP3⁺ Tregs [all p<0.05] whereas proliferation rate was lower than in the CD28⁺ counterparts [50% (0-93.6) non proliferating cells vs. 4.6% (0-30.6), p=0.001]. In contrast, apoptosis induction was higher in CD28^–FoxP3⁺ than in CD28^–FoxP3⁺ Tregs [22.1% (0-30.8) vs. 4.4% (0-7.8), p<0.001]. TCR diversity was also reduced in CD28^–FoxP3⁺ Tregs compared to their CD28⁺ counterparts [median TCR diversity score: 84 (36-104) vs. 115 (109-125), p=0.037].

Conclusion

We discovered a novel T cell subset which combines both senescent as well as regulatory properties. This subset favors the pro-inflammatory milieu and shows altered phenotype and function compared to normal (non-senescent) Tregs.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/altered-phenotype-and-function-of-senescent-regulatory-t-cells-in-rheumatoid-arthritis/