CD28 co-stimulation is intact and contributes to prolongedex vivo survival of hyporesponsive synovial fluid T cells in rheumatoid arthritis

Abstract

In rheumatoid arthritis (RA), T cells in the inflamed joint are considered to play a crucial role in the pathogenesis. However, despite the fact that synovial T cells have an activated memory phenotype, they are functionally suppressed upon combined CD3 and CD28 stimulation. Here, we analyzed the contribution of both CD3 and CD28 to the hyporesponsiveness of synovial T cells in RA. In contrast to the low CD3 responsiveness of synovial fluid (SF) T cells compared to peripheral blood (PB) T cells, the CD28 co-stimulatory response was observed to be unaffected. Hyporesponsiveness of SF T cells has previously been associated with decreased levels of intracellular glutathione (GSH), an antioxidant and regulator of the intracellular redox state. Treatment of SF T cells with N-acetylcysteine, an antioxidant and replenisher of GSH, selectively improved CD3-induced responses, while leaving CD28 responsiveness unaffected. These data show that the CD3 pathway is highly sensitive to intracellular GSH alterations, whereas CD28 responsiveness is relatively refractory. Furthermore, in support for a functional role of CD28 co-stimulation, it was demonstrated that CD28 ligation acted in synergy with the IL-2 receptor gamma chain signaling cytokine IL-15 in the enhancement of the ex vivo survival of SF T cells. These data indicate that CD28 co-stimulatory capacity of SF T cells, in contrast to CD3 stimulation, remains intact despite an altered intracellular redox state. Thereby, CD28 stimulation may contribute to the persistence of T cells at the site of inflammation, which might be of relevance in the pathogenesis of RA.

The Presumed Hyporesponsive Behavior of Rheumatoid Arthritis T Lymphocytes Can Be Attributed to Spontaneous Ex Vivo Apoptosis rather than Defects in T Cell Receptor Signaling

“…Using intracellular FACS staining, it was observed that synovial T cells expressed low levels of antiapoptotic Bcl-2 (41). Under these circumstances, it was proposed that synovial T cell apoptosis in situ was actively suppressed by cell-cell contacts, signaling of IL-2 and IL-15 via the IL-2 receptor common ␥-chain, and/or CD28 costimulation, each of which could enhance Bcl-x L protein expression (41,42,57). In our analysis of mRNA expression in RA SF T cells, we did observe a modest decrease in Bcl-2 expression, but no differences in Bcl-x L expression, compared with HD and RA PB T lymphocytes.…”

“…One possible explanation for the observed discrepancy between RA SF T cell proliferative and cytokine responses in bulk cultures and single-cell analyses could be changes in RA SF T cell viability, as these cells have been reported to quickly undergo apoptosis ex vivo (41,42). Assays using [ 3 H]thymidine incorporation and tissue culture supernatant ELISA analyses cannot accurately account for apoptosis which may occur during extended cell culture.…”

CTLA‐4IG suppresses reactive oxygen species by preventing synovial adherent cell–induced inactivation of rap1, a ras family GTPASE mediator of oxidative stress in rheumatoid arthritis T cells

“…We therefore turned our attention to the possible role of CD28 in this process, since we and other investigators have previously demonstrated that CD28 costimulatory signaling can block T cell Rap1 activation via RapGAP I (27,28). In addition, CD28 signaling pathways, as opposed to those of CD3, have been reported to be intact in RA SF T lymphocytes (29).…”

The CD7− subset of CD4+ memory T cells is prone to accelerated apoptosis that is prevented by interleukin-15 (IL-15)

“…This indicates that 17 and with acute and chronic viral infections 18,19 including HIV, 20,21 in T cells from patients with allogeneic bone 22 and in synovial T cells from joints affected with rheumatoid arthritis. 23,24 Supplementation of Tcell cultures with IL-2R g-chain cytokines upregulates Bcl-2 and Bcl-xL expression and decreases the number of apoptotic cells in vitro implying that cytokine deprivation may be responsible for accelerated apoptosis of these cells. 25,26 Whereas IL-15 is active, IL-2 in concentrations of 1 ± 10 nM, in contrast, fails to rescue CD4 + CD7 7 T cells from apoptosis in vitro.…”